Effects of pulsed electric fields on inactivation kinetics of Listeria innocua

The effects of pulsed electric field (PEF) treatment and processing factors on the inactivation kinetics of Listeria innocua NCTC 11289 were investigated by using a pilot plant PEF unit with a flow rate of 200 liters/h. The electric field strength, pulse length, number of pulses, and inlet temperature were the most significant process factors influencing the inactivation kinetics. Product factors (pH and conductivity) also influenced the inactivation kinetics. In phosphate buffer at pH 4.0 and 0.5 S/m at 40 degrees C, a 3. 0-V/microm PEF treatment at an inlet temperature of 40 degrees C resulted in > or = 6.3 log inactivation of strain NCTC 11289 at 49.5 degrees C. A synergistic effect between temperature and PEF inactivation was also observed. The inactivation obtained with PEF was compared to the inactivation obtained with heat. We found that heat inactivation was less effective than PEF inactivation under similar time and temperature conditions. L. innocua cells which were incubated for a prolonged time in the stationary phase were more resistant to the PEF treatment, indicating that the physiological state of the microorganism plays a role in inactivation by PEF. Sublethal injury of cells was observed after PEF treatment, and the injury was more severe when the level of treatment was increased. Overall, our results indicate that it may be possible to use PEF in future applications in order to produce safe products.     

Effect of High-Pressure-Induced Ice I-to-Ice III Phase Transitions on Inactivation of Listeria innocua in Frozen Suspension

The inactivation of Listeria innocua BGA 3532 at subzero temperatures and pressures up to 400 MPa in buffer solution was studied to examine the impact of high-pressure treatments on bacteria in frozen matrices. The state of aggregation of water was taken into account. The inactivation was progressing rapidly during pressure holding under liquid conditions, whereas in the ice phases, extended pressure holding times had comparatively little effect. The transient phase change of ice I to other ice polymorphs (ice II or ice III) during pressure cycles above 200 MPa resulted in an inactivation of about 3 log cycles, probably due to the mechanical stress associated with the phase transition. This effect was independent of the applied pressure holding time. Flow cytometric analyses supported the assumption of different mechanisms of inactivation of L. innocua in the liquid phase and ice I (large fraction of sublethally damaged cells due to pressure inactivation) in contrast to cells subjected to ice I-to-ice III phase transitions (complete inactivation due to cell rupture). Possible applications of high-pressure-induced phase transitions include cell disintegration for the recovery of intracellular components and inactivation of microorganisms in frozen food.     

Growth of an ice phase in frozen tissue studied by proton NMR-spectroscopy

Development of an ice phase in frozen tissue is a severe problem in cryo-ultramicrotomy. It is assumed that the growth-rate of such an ice phase is controlled by the mobility of the water molecules in the tissue. In order to verify this, wide-line nmr-spectroscopy has been performed on the protons of frozen whole blood, with and without protective agent, in the temperature range −100 °C- −40 °C. The molecular motion may be characterized by a correlation time, τ, which can be thought of as roughly the mean time between proton jumps. The value of τ can be approximately evaluated from the width of the proton resonance line. It is shown that τ varies rapidly with temperature, in a similar manner to maximum storage time for frozen blood in blood banks. Addition of glycerol increases τ at low temperature, which is consistent with its use as a protective agent in cryobiology. At high temperature, addition of glycerol reduces τ, which is in agreement with reported light-microscope observations on the growth-rate of an ice phase in the same temperature range, nmr-spectroscopy seems to be a useful tool in the further development of cryo-ultramicrotomy. However, to estimate the value in full, more data is required than is presented here.     

Effects of Pulsed Electric Fields on Inactivation Kinetics of Listeria innocua

The effects of pulsed electric field (PEF) treatment and processing factors on the inactivation kinetics of Listeria innocua NCTC 11289 were investigated by using a pilot plant PEF unit with a flow rate of 200 liters/h. The electric field strength, pulse length, number of pulses, and inlet temperature were the most significant process factors influencing the inactivation kinetics. Product factors (pH and conductivity) also influenced the inactivation kinetics. In phosphate buffer at pH 4.0 and 0.5 S/m at 40°C, a 3.0-V/μm PEF treatment at an inlet temperature of 40°C resulted in ≥6.3 log inactivation of strain NCTC 11289 at 49.5°C. A synergistic effect between temperature and PEF inactivation was also observed. The inactivation obtained with PEF was compared to the inactivation obtained with heat. We found that heat inactivation was less effective than PEF inactivation under similar time and temperature conditions. L. innocua cells which were incubated for a prolonged time in the stationary phase were more resistant to the PEF treatment, indicating that the physiological state of the microorganism plays a role in inactivation by PEF. Sublethal injury of cells was observed after PEF treatment, and the injury was more severe when the level of treatment was increased. Overall, our results indicate that it may be possible to use PEF in future applications in order to produce safe products.     

Effect of suspension media on nonthermal inactivation of Escherichia coli

AIMS: To investigate the influence of suspension media on the survival of Escherichia coli M23 exposed to nonthermal, lethal stresses. METHODS AND RESULTS: Populations of E. coli M23 suspended in minimal medium (MM) or in different nutrient-rich broths were exposed to water activity 0.90 and/or pH 3.5 and inactivation was determined by culture-based enumeration. In response to the osmotic or acid challenges, E. coli M23 displayed enhanced survival in MM rather than in complex broth. That trend was reversed when populations were exposed to low water activity in combination with low pH. Comparison of microbial survival in three complex media indicated that even relatively small differences in composition influenced inactivation. In most media the combination of lethal stresses resulted in a synergism, which enhanced bacterial inactivation; however, an exception (tryptone soya broth) was observed. CONCLUSIONS: The suspension medium strongly influences the inactivation of E. coli M23 by osmotic and/or acid stresses. This should be considered when comparing studies of microbial survival that use different media and when broth-derived data are intended to represent specific environments (e.g. food matrices). SIGNIFICANCE AND IMPACT OF THE STUDY: The specific effects of synthetic media need to be appreciated when studying bacterial inactivation in conditions relevant to food-manufacturing regimes.     

Survival of Listeria innocua on hot and cold beef carcass surfaces

AIMS: This study aimed to determine the survival and growth of Listeria innocua on hot and cold beef carcass surfaces. METHODS AND RESULTS: Four sites, the neck, outside round, brisket and foreshank/brisket, were inoculated with L. innocua (i) immediately after dressing while hot and (ii) when cold after chilling. After inoculation, all carcasses were stored at 4 degrees C for 72 h. Survival of L. innocua on cold surfaces declined during storage and was less than on hot carcasses at all times. Data on the survival of L. innocua in broth (maximum recovery diluent) indicated that counts could not be compared with those on carcasses, in particular on cold carcasses. CONCLUSIONS: The results indicate that L. innocua survives on hot carcass surfaces during chilling, but declines over time on cold surfaces. The decrease in L. innocua counts on cold surfaces may be related to a synergy between the combined stresses of low available water (a(w)) and low temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to determine the effect of chilling on the survival and growth of Listeria on beef carcass surfaces. The information can potentially be used to determine the survival and growth of the pathogen, L. monocytogenes on beef surfaces.     

Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua

Aims: Detectability of Listeria monocytogenes at 10⁰ CFU per food sample in the presence of Listeria innocua using standard microbiological detection was evaluated and compared with the real‐time PCR‐based method. Methods and Results: Enrichment in half‐Fraser broth followed by subculture in Fraser broth according to EN ISO 11290‐1 was used. False‐negative detection of 10⁰ CFU L. monocytogenes was obtained in the presence of 10¹ CFU L. innocua per sample using the standard detection method in contrast to more than 10⁵ CFU L. innocua per sample using real‐time PCR. Identification of L. monocytogenes on the chromogenic medium by the standard procedure was impossible if L. innocua was able to overgrow L. monocytogenes by more than three orders of magnitude after the enrichment in model samples. These results were confirmed using naturally contaminated food samples. Conclusions: Standard microbiological method was insufficient for the reliable detection of 10⁰ CFU L. monocytogenes in the presence of more than 10⁰ CFU of L. innocua per sample. On the other hand, if the growth of L. monocytogenes was sufficient to reach the concentration equal to the detection limit of PCR, the amount of the other microflora present in the food sample including L. innocua was not relevant for success of the PCR detection of L. monocytogenes. Significance and Impact of the Study: After the enrichment, the PCR detection is more convenient than the standard one as PCR detection is not compromised by other present microflora.     

Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua

A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism.     

The effect of temperature on culturability and detection of Listeria innocua in water

The survival of Listeria innocua in culturable form in water is temperature-dependent. Incubation at 30°C results in a rapid loss of culturability. DNA from Listeria innocua can be detected by PCR amplification in cells which are no longer culturable.     

Effect of inorganic cations on phase transitions.

The effect of protons and cations on the crystal (gel)-to-liquid crystal transition temperature Tm of isoelectric and negatively charged phospholipids are summarized. The general trends emerging are as follows: Tm depends on the state of ionization of the phospholipid in that Tm-vs-pH-curves parallel the titration curve of the phospholipid. Protonation of phospholipids causes Tm to increase, deprotonation or ionization has the opposite effect. The effects of cations on the Tm of phospholipids may be grouped into non-specific and specific effects. Unspecific effects of cations such as the screening of negative charges of the phospholipid polar group are qualitatively similar to protonation: Tm increases, in the order monovalent less than divalent less than trivalent cations and the effects on negatively charged phospholipids are larger than those on isoelectric phospholipids. Unspecific, electrostatic effects on Tm are reasonably well accounted for by the Gouy-Chapman theory. If, however, specific binding comes into play and/or electrostatic effects are accompanied by changes in phospholipid structure, simple, electrostatic theories fail to explain the observed changes in Tm. The crystal (gel)-to-liquid crystal transition is also a function of the degree of hydration: Tm generally decreases with increasing hydration reaching a plateau in excess H2O. In addition to screening of electric charges, ions may exert yet another non-specific effect: ions may affect Tm indirectly by competing with the phospholipid polar group for water of hydration. This indirect effect plays a role at high ionic strength and/or at low hydration of the phospholipid. Specific binding of cations to negatively charged phospholipids can lead to tight associations of the metal ion with the lipid polar group. Isothermal crystallization of the phospholipid bilayer is induced that is accompanied by a total or partial loss of water of hydration resulting in a marked increase in Tm. For instance, in crystalline Ca2(+)-phosphatidylserine complexes Tm is increased by more than 100 degrees C.     

An alternative explanation for phase transitions observed in quick frozen calcium cardiolipin solution

It is demonstrated that the phase changes reported for a quick frozen calcium cardiolipin solution containing CaCl₂ are virtually identical to those seen in pure CaCl₂. This introduces uncertainty as to whether the data in fact reflect the behavior of cardiolipin or of the associated CaCl₂.     

Relationship between inactivation kinetics of a Listeria monocytogenes suspension by chlorine and its chlorine demand

AIMS: Chlorine demand by Listeria monocytogenes cells and inactivation of L. monocytogenes by chlorine (0.6-1.0 mg l(-1)) at different temperatures (4, 20 and 30 degrees C) have been investigated in a batch reactor. METHODS AND RESULTS: Chlorine demand depended on the microbial concentration and was independent on the initial chlorine concentration and temperature. Chlorine decay was modelled by the addition of two first-order decay equations. Inactivation of L. monocytogenes by chlorine depended on the initial microbial concentration, initial chlorine concentration and temperature. A mathematical model based on a biphasic inactivation properly described survival curves of L. monocytogenes and a tertiary model was developed that satisfactorily predicted the inactivation of L. monocytogenes by different concentrations of initial chlorine at different temperatures. CONCLUSIONS: Both available chlorine decay and inactivation of L. monocytogenes by chlorine were biphasic and can be modelled by a two-term exponential model. SIGNIFICANCE AND IMPACT OF THE STUDY: The biphasic nature of survival curves of L. monocytogenes did not reflect the effect of a change of available chlorine concentration during the treatment. The microbial inactivation was caused by successive reactions that occur after the consumption of the chlorine by the bacterial cell components.     

Effect of alcohols on the phase transitions of dihexadecylphosphatidylcholine

We have systematically investigated the effect of short chain alcohols (methanol to n-propanol) on the phase transitions of 1,2-dihexadecylphosphatidylcholine (DHPC), a lipid that forms a stable interdigitated gel phase (L beta I) in aqueous solution. The temperature of the low-temperature L beta I to P beta' phase transition of DHPC was found to increase with alcohol concentration, showing that alcohol interacts preferentially with the interdigitated phase relative to the non-interdigitated gel. The main transition of DHPC exhibited a biphasic effect of alcohol concentration similar to that previously observed with DPPC (Rowe, E.S. (1983) Biochemistry 22,3299-3305). As alcohol concentration is increased the lower L beta I to P beta' and main P beta' to L alpha transitions of DHPC merge at the threshold concentration of the biphasic effect, so that above this concentration there is one phase transition from L beta I directly to L alpha. This is analogous to DPPC above its biphasic threshold. Similar to DPPC, the transition between L beta I and L alpha exhibits marked hysteresis.     

Genetic basis of tetracycline resistance in food-borne isolates of Listeria innocua.

Eleven of 12 tetracycline-resistant Listeria innocua strains, isolated from chicken or turkey frankfurters and mozzarella cheese, were shown to carry DNA sequences which hybridized with the Tet M probe; of these, two strains also hybridized with Tet K. The remaining strain hybridized with the Tet K probe only. The Tet M determinant appeared to be located on the chromosome; in one case, it was transferable by conjugation to recipients Listeria monocytogenes, Listeria ivanovii, and Enterococcus faecalis.