Exposure to salt and organic acids increases the ability of Listeria monocytogenes to invade Caco-2 cells but decreases its ability to survive gastric stress

The effects of environmental stress exposure on Listeria monocytogenes growth and virulence-associated characteristics were investigated. Specifically, we measured the effects of temperature (7 or 37 degrees C), pH (5.5 or 7.4), the presence of salt and organic acids (375 mM NaCl, 8.45 mM sodium diacetate [SD], 275 mM sodium lactate [SL], or a combination of NaCl, SD, and SL), and deletion of sigB, which encodes a key stress response regulator, on the ability of L. monocytogenes to grow, invade Caco-2 cells, and survive exposure to synthetic gastric fluid (pH 2.5 or 4.5). Our results indicate that (i) L. monocytogenes log-phase generation times and maximum cell numbers are not dependent on the alternative sigma factor sigmaB in the presence of NaCl and organic acids at concentrations typically found in foods; (ii) growth inhibition of L. monocytogenes through the addition of organic acids is pH dependent; (iii) the ability of L. monocytogenes to invade Caco-2 cells is affected by growth phase, temperature, and the presence of salt and organic acids, with the highest relative invasion capabilities observed for cells grown with SL or NaCl at 37 degrees C and pH 7.4; (iv) growth of L. monocytogenes in the presence of NaCl, SD, or SL reduces its ability to survive exposure to gastric fluid; and (v) exposure of L. monocytogenes to gastric fluid reduces the enhanced invasiveness caused by growth in the presence of NaCl or SL. These findings suggest that virulence-associated characteristics that determine the L. monocytogenes infectious dose are likely to be affected by food-specific properties (e.g., pH or the presence of salt or organic acid).     

Role of Listeria monocytogenes σB in Survival of Lethal Acidic Conditions and in the Acquired Acid Tolerance Response

The food-borne pathogen Listeria monocytogenes can acquire enhanced resistance to lethal acid conditions through multiple mechanisms. We investigated contributions of the stress-responsive alternative sigma factor, σ^B, which is encoded by sigB, to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in L. monocytogenes. At various points throughout growth, we compared the relative survival of L. monocytogenes wild-type and ΔsigB strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation. Under these conditions, survival of the ΔsigB strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase. Survival of both ΔsigB and wild-type L. monocytogenes strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the L. monocytogenes growth phase-dependent AR mechanism is σ^B independent. σ^B-mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth. Loss of a functional σ^B reduced the survival of L. monocytogenes at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that L. monocytogenes protection against organic and inorganic acid may be mediated through different mechanisms. σ^B does not appear to contribute to pH_i homeostasis through regulation of net proton movement across the cell membrane or by regulation of pH_i buffering by the GAD system under the conditions examined in this study. In summary, a functional σ^B protein is necessary for full resistance of L. monocytogenes to lethal acid treatments.     

Effect of Food Processing-Related Stresses on Acid Tolerance of Listeria monocytogenes

Stationary-phase cells of Listeria monocytogenes grown in glucose-free or glucose-containing media were exposed for 90 min to various stresses, including acid stress (pH 4.0 to 7.0), osmotic stress (10.5 to 20.5% NaCl), and various temperatures (−5 to 50°C), and were further exposed to pH 3.5. Exposure to a mildly acidic (pH 5.0 to 6.0) environment provided protection of the pathogen against acid upon subsequent exposure. This adaptive response, however, was found to be strongly dependent on other environmental conditions during the shock, such as temperature or the simultaneous presence of a second stress factor (NaCl). Growth of L. monocytogenes in the presence of glucose resulted in enhanced survival of the pathogen at pH 3.5. Sublethal stresses other than acidic stresses, i.e., osmotic, heat, and low-temperature stresses, did not affect the acid resistance of L. monocytogenes (P > 0.5). More-severe levels of these stresses, however, resulted in sensitization of the pathogen to acid.     

Ability of the Listeria monocytogenes Strain Scott A To Cause Systemic Infection in Mice Infected by the Intragastric Route

Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involve Listeria monocytogenes serotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates of L. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection with L. monocytogenes Scott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strain L. monocytogenes EGD. Prior administration of sodium bicarbonate did not enhance the virulence of L. monocytogenes strain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains of L. monocytogenes (Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth of L. monocytogenes Scott A at temperatures of 12.5 to 37°C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4b L. monocytogenes strains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.     

Identification and Characterization of Di- and Tripeptide Transporter DtpT of Listeria monocytogenes EGD-e

Listeria monocytogenes is a gram-positive intracellular pathogen responsible for opportunistic infections in humans and animals. Here we identified and characterized the dtpT gene (lmo0555) of L. monocytogenes EGD-e, encoding the di- and tripeptide transporter, and assessed its role in growth under various environmental conditions as well as in the virulence of L. monocytogenes. Uptake of the dipeptide Pro-[¹⁴C]Ala was mediated by the DtpT transporter and was abrogated in a ΔdtpT isogenic deletion mutant. The DtpT transporter was shown to be required for growth when the essential amino acids leucine and valine were supplied as peptides. The protective effect of glycine- and proline-containing peptides during growth in defined medium containing 3% NaCl was noted only in L. monocytogenes EGD-e, not in the ΔdtpT mutant strain, indicating that the DtpT transporter is involved in salt stress protection. Infection studies showed that DtpT contributes to pathogenesis in a mouse infection model but has no role in bacterial growth following infection of J774 macrophages. These studies reveal that DptT may contribute to the virulence of L. monocytogenes.     

The growth limits of a large number of Listeria monocytogenes strains at combinations of stresses show serotype--and niche-specific traits

Aims: The aim of this study was to associate the growth limits of Listeria monocytogenes during exposure to combined stresses with specific serotypes or origins of isolation, and identify potential genetic markers. Methods and Results: The growth of 138 strains was assessed at different temperatures using combinations of low pH, sodium lactate, and high salt concentrations in brain heart infusion broth. None of the strains was able to grow at pH ≤ 4·4, a_w ≤ 0·92, or pH ≤ 5·0 combined with a_w ≤ 0·94. In addition, none of the strains grew at pH ≤ 5·2 and NaLac ≥ 2%. At 30°C, the serotype 4b strains showed the highest tolerance to low pH and high NaCl concentrations at both pH neutral (pH 7·4) and mild acidic conditions (pH 5·5). At 7°C, the serotype 1/2b strains showed the highest tolerance to high NaCl concentrations at both pH 7·4 and 5·5. Serotype 1/2b meat isolates showed the highest tolerance to low pH in the presence of 2% sodium lactate at 7°C. ORF2110 and gadD1T1 were identified as potential biomarkers for phenotypic differences. Conclusions: Differences in growth limits were identified between specific L. monocytogenes strains and serotypes, which could in some cases be associated with specific genetic markers. Significance and Impact of the Study: Our data confirm the growth limits of L. monocytogenes as set out by the European Union for ready-to-eat foods and provides an additional criterion. The association of L. monocytogenes serotypes with certain stress responses might explain the abundance of certain serotypes in retail foods while others are common in clinical cases.     

Modeling the Growth of Listeria monocytogenes in Soft Blue-White Cheese

The aim of this study was to develop a predictive model simulating growth over time of the pathogenic bacterium Listeria monocytogenes in a soft blue-white cheese. The physicochemical properties in a matrix such as cheese are essential controlling factors influencing the growth of L. monocytogenes. We developed a predictive tertiary model of the bacterial growth of L. monocytogenes as a function of temperature, pH, NaCl, and lactic acid. We measured the variations over time of the physicochemical properties in the cheese. Our predictive model was developed based on broth data produced in previous studies. New growth data sets were produced to independently calibrate and validate the developed model. A characteristic of this tertiary model is that it handles dynamic growth conditions described in time series of temperature, pH, NaCl, and lactic acid. Supplying the model with realistic production and retail conditions showed that the number of L. monocytogenes cells increases 3 to 3.5 log within the shelf life of the cheese.     

Growth of Halotolerant Food Spoiling Yeast Debaryomyces nepalensis NCYC 3413 Under the Influence of pH and Salt

Debaryomyces nepalensis, a halotolerant food-spoiling yeast could grow in complex (YEPD) medium at different pHs ranging between 3.0 and 11.0 in the absence of salt and at pH 3.0–9.0 in the presence of different concentrations of NaCl and KCl. The specific growth rate of D. nepalensis was not affected by the initial pH of the medium in the absence of salts, whereas it was affected in the presence of salts. At 2 M NaCl and KCl, the organism exhibited a synergistic effect on pH and salt stress, which was unique in the Debaryomyces species. Irrespective of the initial pH and salt, the intracellular pH of D. nepalensis was ~7.0. Significant organic acid was produced at neutral and alkaline pH and organic acid production increased with the increase in pH and salt. Very specific organic acids are produced in the presence of NaCl and KCl. Our observation would contribute to a better understanding of the physiological phenomenon of halotolerance in D. nepalensis.     

Relationship between Sodium Influx and Salt Tolerance of Nitrogen-Fixing Cyanobacteria

The relationship between sodium uptake and cyanobacterial salt (NaCl) tolerance has been examined in two filamentous, heterocystous, nitrogen-fixing species of Anabaena. During diazotrophic growth at neutral pH of the growth medium, Anabaena sp. strain L-31, a freshwater strain, showed threefold higher uptake of Na⁺ than Anabaena torulosa, a brackish-water strain, and was considerably less salt tolerant (50% lethal dose of NaCl, 55 mM) than the latter (50% lethal dose of NaCl, 170 mM). Alkaline pH or excess K⁺ (>25 mM) in the medium causes membrane depolarization and inhibits Na⁺ influx in both cyanobacteria (S. K. Apte and J. Thomas, Eur. J. Biochem. 154:395-401, 1986). The presence of nitrate or ammonium in the medium caused inhibition of Na⁺ influx accompanied by membrane depolarization. These experimental manipulations affecting Na⁺ uptake demonstrated a good negative correlation between Na⁺ influx and salt tolerance. All treatments which inhibited Na⁺ influx (such as alkaline pH, K⁺ above 25 mM, NO₃⁻, and NH₄⁺), enhanced salt tolerance of not only the brackish-water but also the freshwater cyanobacterium. The results indicate that curtailment of Na⁺ influx, whether inherent or effected by certain environmental factors (e.g., combined nitrogen, alkaline pH), is a major mechanism of salt tolerance in cyanobacteria.     

Salt tolerance mechanisms in three Irano-Turanian Brassicaceae halophytes relatives of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Salt tolerance mechanisms were studied in three Irano-Turanian halophytic species from the Brassicaceae ??(Lepidium latifolium, L. perfoliatum and Schrenkiella parvula) and compared with the glycophyte Arabidopsis thaliana. According to seed germination under salt stress, L. perfoliatum was the most tolerant species, while L. latifolium and S. parvula were rather susceptible. Contrastingly, based on biomass production L. perfoliatum was more salt sensitive than the other two species. In S. parvula biomass was increased up to 2.8-fold by 100 mM NaCl; no significant growth reduction was observed even when exposed to 400 mM NaCl. Stable activities of antioxidative defense enzymes, nil or negligible accumulation of superoxide anion and hydrogen peroxide, as well as stable membrane integrity in the three halophytes revealed that no oxidative stress occurred in these tolerant species under salt stress. Proline levels increased in response to salt treatment. However, it contributed only by 0.3?2.0% to the total osmolyte concentration in the three halophytes (at 400 mM NaCl) and even less (0.04%) in the glycophyte, A. thaliana (at 100 mM NaCl). Soluble sugars in all three halophytes and free amino acids pool in S. parvula decreased under salt treatment in contrast to the glycophyte, A. thaliana. The contribution of organic osmolytes to the total osmolyte pool increased by salt treatment in the roots, while decreased in halophyte and glycophyte, A. thaliana leaves. Interestingly, this reduction was compensated by a higher relative contribution of K in the leaves of the halophytes, but of Na in A. thaliana. Taken together, biomass data and biochemical indicators show that S. parvula is more salt tolerant than the two Lepidium species. Our data indicate that L. latifolium, as a perennial halophyte with a large biomass, is highly suitable for both restoration of saline habitats and saline agriculture.     

Sensitization of Listeria monocytogenes to Low pH, Organic Acids, and Osmotic Stress by Ethanol

The killing of Listeria monocytogenes following exposure to low pH, organic acids, and osmotic stress was enhanced by the addition of 5% (vol/vol) ethanol. At pH 3, for example, the presence of this agent stimulated killing by more than 3 log units in 40 min of exposure. The rate of cell death at pH 3.0 was dependent on the concentration of ethanol. Thus, while the presence 10% (vol/vol) ethanol at pH 3.0 stimulated killing by more than 3 log units in just 5 min, addition of 1.25% (vol/vol) ethanol resulted in less than 1 log unit of killing in 10 min. The ability of 5% (vol/vol) ethanol to stimulate killing at low pH and at elevated osmolarity was also dependent on the amplitude of the imposed stress, and an increase in the pH from 3.0 to 4.0 or a decrease in the sodium chloride concentration from 25 to 2.5% led to a marked reduction in the effectiveness of 5% (vol/vol) ethanol as an augmentative agent. Combinations of organic acids, low pH, and ethanol proved to be particularly effective bactericidal treatments; the most potent combination was pH 3.0, 50 mM formate, and 5 % (vol/vol) ethanol, which resulted in 5 log units of killing in just 4 min. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane.     

Effect of Bacteriocins and Conditions that Mimic Food and Digestive Tract on Biofilm Formation, In Vitro Invasion of Eukaryotic Cells and Internalin Gene Expression by Listeria monocytogenes

In this report, Listeria monocytogenes isolates were evaluated for their ability to form biofilms, for adhesion/invasion of eukaryotic cells and for differential expression of internalin A (inl A) gene, which is related to virulence potential. The presence of bacteriocins of lactic acid bacteria and incubation at 5 °C were the main factors that influenced biofilm formation by L. monocytogenes, in comparison with BHI (control). In general, adhesion and invasion of Caco-2 cells were significantly lower in low pH (4.5), in incubation at 5 °C and in the presence of Oxgall 0.3 %. On the other hand, two L. monocytogenes isolates (INCQS 353 and Reg 26c) showed higher invasion rates when cultivated in the presence of NaCl 5 % (P < 0.05). One L. monocytogenes isolate (H-2) showed the strongest ability to form biofilm and to invade Caco-2 cells, under selected conditions, suggesting there is a relationship between biofilm formation and virulence potential. For all isolates, expression of inl A gene was down-regulated by the presence of bacteriocins, Oxgall 0.3 %, pH 4.5 and incubation at 5 °C. Nonetheless, for one L. monocytogenes isolate (HU 471), expression of inl A gene was eight times higher in the presence of sucrose, indicating that food components can increase the infectiveness of L. monocytogenes.     

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

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.Listeria monocytogenes is a food-borne, human pathogen that has a remarkable ability to colonize food-processing environments (5, 16, 20, 21, 26, 29). Some L. monocytogenes strains can persist for years in food-processing plants (11, 14, 20, 27), and specific molecular subtypes can repeatedly be isolated from the processing environment (29) despite being very infrequent in the outdoor environment (9). This ability to persist has, hitherto, not been linked to any specific genetic or phenotypic trait.It has been suggested that persistent L. monocytogenes strains may be more tolerant or resistant to cleaning and especially disinfectants used in the food industry. Aase et al. (1) found increased tolerance to both benzalkonium chloride and ethidium bromide in L. monocytogenes isolates that had persisted for more than 4 years; however, other studies have not been able to link persistence and tolerance to disinfectants (6, 10, 11, 13). We recently compared disinfection sensitivities of persistent and presumed nonpersistent L. monocytogenes strains using viable-cell counts and did not find the latter group more sensitive to the two disinfectants Triquart SUPER and Incimaxx DES than persistent strains (13). However, we found that for all subtypes of L. monocytogenes, growth with NaCl increased the tolerance of planktonic L. monocytogenes cells to Incimaxx DES, whereas spot-inoculated, dried L. monocytogenes cells were not protected by NaCl against disinfection.There is no doubt that L. monocytogenes will be completely inactivated at the disinfectant concentrations recommended for use in the food industry; however, the efficiency of the disinfectant is very much influenced by the presence of organic material being inactivated by the presence of food debris. Hence, it is likely that the bacterial cell in a food production environment may be exposed to concentrations at a sublethal level. It is currently not known if treatment with a sublethal concentration of disinfectant affects the entire bacterial population or only attacks a fraction of the cell population, leaving another fraction of cells unaffected. In case of the latter, some bacterial cells may be able to survive the disinfection treatment. The potential presence of such tolerant subpopulations could, ultimately, ensure that the genome is propagated, leading to persistence.The presence of a more tolerant subpopulation can be determined on the single-cell level. Flow cytometry is a rapid method useable for measurements at the single-cell resolution (22); however, it cannot monitor the same single cells over time. Optical microscopy combined with microfluidic devices that allow measurement of growth of single cells is a useful technique (2), and in situ analyses of the physiological condition of single cells by the fluorescence ratio imaging microscopy (FRIM) technique represents another elegant approach (25). FRIM enables studies of dynamic changes with high sensitivity and on the single-cell level in important physiological parameters: e.g., intracellular pH (pH_i). Listeria maintains its pH_i within a narrow range of 7.6 to 8 at extracellular pH (pH_ex) values of 5.0 to 8.0 (4, 25) and at pH_ex 4.0 with the presence of glucose (23). It is believed that viable cells need to maintain a transmembrane pH gradient with their pH_i above the pH_ex, and failure to maintain pH_i homeostasis indicates that the bacterial cell is severely stressed and ultimately leads to loss of cell viability. FRIM has been used to determine the pH_i of L. monocytogenes after exposure to osmotic and acid stress (7, 23). Also, the dissipation of the pH gradient in L. monocytogenes after exposure to different bacteriocins has been determined with FRIM (4, 12). Hornbæk 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 (ΔpH) and the other consisting of cells that maintained ΔpH, which could indicate phenotypic heterogeneity.The aim of the present study was to investigate the physiological effects of the disinfectant Incimaxx DES at sublethal and lethal concentrations on single cells and the population level of a persistent L. monocytogenes strain to study a possible subdivision of sensitivity in the population. We also addressed the potential protective effect of NaCl against disinfection and compared sensitivities in a population of planktonic and attached bacteria. We applied the in situ technique FRIM and compared the pH_i measurements with the traditional viable-cell-count method.(Part of the results have been presented at a poster session at the 95th International Association for Food Protection annual meeting in Columbus, OH, 3 to 6 August 2008.)     

Effects of short- and long-term salinity on leaf water relations, gas exchange, and growth in Ipomoea pes-caprae

Ipomoea pes-caprae is widespread in pantropical coastal areas along the beach. The aim of this study was to investigate the salinity tolerance level and physiological mechanisms that allow I. pes-caprae to endure abrupt increases in salinity under brief or prolonged exposure to salinity variations. Xylem sap osmolality (X_osm), leaf water relations, gas exchange, and number of produced and dead leaves were measured at short- (1-7 d) and long- (22-46 d) term after a sudden increase in soil salinity of 0, 85, 170, and 255 mM NaCl. In the short-term, X_osm was not affected by salinity, but in the long-term there was a significant increase in plants grown in presence of salt compared with control plants. After salt addition, the plants showed osmotic stress with temporal cell turgor loss. However, the water potential gradient for water uptake was re-established at 4, 7 and 22 d after salt addition, at 85, 170 and 255 mM NaCl, respectively. In the short-term I. pes-caprae was able to tolerate salinities of up to 255 mM NaCl without significant reduction in carbon assimilation or growth. With the duration of stress, leaf ion concentration continued to increase and reached toxic levels at high salinity with a progressive decrease in photosynthetic rate, reduced leaf formation and accelerated senescence. Then, if high levels of soil salts from tidal inundation occur for short periods, the survival of I. pes-caprae is possible, but prolonged exposure to salinity may induce metabolic damage and reduce drastically the plant growth.     

Effect of NaCl and Polyamines on Plasma Membrane Lipids of Wheat Roots

Caryopses of a salt sensitive wheat cultivar (Triticum aestivum L. cv. Giza 163) were presoaked in 2.5 mM putrescine (Put), 5 mM spermidine (Spd) or 2.5 mM spermine (Spm) for 24 h and then subjected to 150 mM NaCl added to the growth medium for 15 d. Effects of NaCl and polyamines (PAs) on plasma membrane (PM) lipids, phospholipids, fatty acids, and free sterols were determined. NaCl treatment caused a decrease in total phospholipids, increase in saturated fatty acids and altered distribution of sterols and phospholipids. NaCl also induced increase in sterol/phospholipid ratio. PAs treatments (particularly Put and Spd) counterbalanced the NaCl deleterious effects on PM lipids.     

Selection and Identification of a Listeria monocytogenes Target Strain for Pulsed Electric Field Process Optimization

Nine Listeria monocytogenes strains were treated individually with a continuous pulsed electric field (PEF) apparatus, and their sensitivities to the treatment were compared at 25 kV/cm. When cell suspensions of these strains in 0.1% NaCl (pH 7.0) were treated at 23°C for 144 μs, inactivation ranged from 0.7 to 3.7 log₁₀ CFU/ml. Inactivation by 72-μs PEF treatments at 37°C ranged from 0.3 to 2.5 log₁₀ CFU/ml. L. monocytogenes OSY-8578 was substantially more resistant than other strains when cells were PEF treated in 0.1% NaCl, whereas Scott A was one of the most sensitive strains. The superiority of OSY-8578's resistance to that of Scott A was confirmed in 50% diluted acid whey (pH 4.2). Changes in sensitivity to PEF during phases of growth were minimal in OSY-8578 and substantial in Scott A. Use of L. monocytogenes OSY-8578, therefore, is recommended in studies to optimize PEF processes that target L. monocytogenes. The nine L. monocytogenes strains were genotyped with pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) techniques. These strains were better differentiated with PFGE than with AP-PCR. The target strain (OSY-8578) was characterized by both molecular typing techniques, but resistance to PEF, in general, was not associated with a particular genotype group.