High Phenotypic Variability among Representative Strains of Common Salmonella enterica Serovars with Possible Implications for Food Safety.

Salmonella is an important foodborne pathogen, whose ability to resist stress and survive can vary among strains. This variability is normally not taken into account when predictions are made about survival in foods with negative consequences. Therefore, we examined the contribution of variable phenotypic properties to survival under stress in 10 Salmonella serovars. One strain (Typhimurium 10) was intentionally RpoS-negative; however, another strain (Heidelberg) showed an rpoS mutation, rendering it inactive. We assessed an array of characteristics (motility, biofilm formation, bile resistance, acid resistance, and colony morphology) that show major variability among strains associated with a 10- to 19-fold difference between the highest and the lowest strain for most characteristics. The RpoS status of isolates did not affect variability in the characteristics, with the exception of resistance to NaCl, acetic acid, lactic acid, and the combination of acetic acid and salt, where the variability between the highest and the lowest strain was reduced to 3.1-fold, 1.7-fold, 2-fold, and 1.7-fold, respectively, showing that variability was significant among RpoS-positive strains. Furthermore, we also found a good correlation between acid resistance and lysine decarboxylase activity, showing its importance for acid resistance, and demonstrated a possible role of RpoS in the lysine decarboxylase activity in Salmonella.

Synergistic combinations of high hydrostatic pressure and essential oils or their constituents and their use in preservation of fruit juices.

This work addresses the inactivation achieved with Escherichia coli O157:H7 and Listeria monocytogenes EGD-e by combined processes of high hydrostatic pressure (HHP) and essential oils (EOs) or their chemical constituents (CCs). HHP treatments (175-400 MPa for 20 min) were combined with 200 µL/L of each EO (Citrus sinensis L., Citrus lemon L., Citrus reticulata L., Thymus algeriensis L., Eucalyptus globulus L., Rosmarinus officinalis L., Mentha pulegium L., Juniperus phoenicea L., and Cyperus longus L.) or each CC ((+)-limonene, α-pinene, ß-pinene, p-cymene, thymol, carvacrol, borneol, linalool, terpinen-4-ol, 1,8-cineole, α-terpinyl acetate, camphor, and (+)-pulegone) in buffer of pH 4.0 or 7.0. The tested combinations achieved different degrees of inactivation, the most effective being (+)-limonene, carvacrol, C. reticulata L. EO, T. algeriensis L. EO and C. sinensis L. EO which were capable of inactivating about 4-5 log(10) cycles of the initial cell populations in combination with HHP, and therefore showed outstanding synergistic effects. (+)-Limonene was also capable of inactivating 5 log(10) cycles of the initial E. coli O157:H7 population in combination with HHP (300 MPa for 20 min) in orange and apple juices, and a direct relationship was established between the inactivation degree caused by the combined process with (+)-limonene and the occurrence of sublethal injury after the HHP treatment. This work shows the potential of EOs and CCs in the inactivation of foodborne pathogens in combined treatments with HHP, and proposes their possible use in liquid food such as fruit juices.

The inhibitory effect of natural microflora of food on growth of Listeria monocytogenes in enrichment broths.

The aims of this study were to (i) compare the inhibitory effects of the natural microflora of different foods on the growth of Listeria monocytogenes during enrichment in selective and non-selective broths; (ii) to isolate and identify components of the microflora of the most inhibitory food; and (iii) to determine which of these components was most inhibitory to growth of L. monocytogenes in co-culture studies. Growth of an antibiotic-resistant marker strain of L. monocytogenes was examined during enrichment of a range of different foods in Tryptone Soya Broth (TSB), Half Fraser Broth (HFB) and Oxoid Novel Enrichment (ONE) Broth. Inhibition of L. monocytogenes was greatest in the presence of minced beef, salami and soft cheese and least with prepared fresh salad and chicken pâté. For any particular food the numbers of L. monocytogenes present after 24h enrichment in different broths increased in the order: TSB, HFB and ONE Broth. Numbers of L. monocytogenes recovered after enrichment in TSB were inversely related to the initial aerobic plate count (APC) in the food but with only a moderate coefficient of determination (R(2)) of 0.51 implying that microbial numbers and the composition of the microflora both influenced the degree of inhibition of L. monocytogenes. In HFB and ONE Broth the relationship between APC and final L. monocytogenes counts was weaker. The microflora of TSB after 24h enrichment of minced beef consisted of lactic acid bacteria, Brochothrix thermosphacta, Pseudomonas spp., Enterobacteriaceae, and enterococci. In co-culture studies of L. monocytogenes with different components of the microflora in TSB, the lactic acid bacteria were the most inhibitory followed by the Enterobacteriaceae. The least inhibitory organisms were Pseudomonas sp., enterococci and B. thermosphacta. In HFB and ONE Broth the growth of Gram-negative organisms was inhibited but lactic acid bacteria still reached high numbers after 24h. A more detailed study of the growth of low numbers of L. monocytogenes during enrichment of minced beef in TSB revealed that growth of L. monocytogenes ceased at a cell concentration of about 10(2)cfu/ml when lactic acid bacteria entered stationary phase. However in ONE Broth growth of lactic acid bacteria was slower than in TSB with a longer lag time allowing L. monocytogenes to achieve much higher numbers before lactic acid bacteria reached stationary phase. This work has identified the relative inhibitory effects of different components of a natural food microflora and shown that the ability of low numbers of L. monocytogenes to achieve high cell concentrations is highly dependent on the extent to which enrichment media are able to inhibit or delay growth of the more effective competitors.

The relationship between membrane damage, release of protein and loss of viability in Escherichia coli exposed to high hydrostatic pressure.

The aim of this work was to examine a possible association between resistance of two Escherichia coli strains to high hydrostatic pressure and the susceptibility of their cell membranes to pressure-induced damage. Cells were exposed to pressures between 100 and 700 MPa at room temperature (approximately 20 degrees C) in phosphate-buffered-saline. In the more pressure-sensitive strain E. coli 8164, loss of viability occurred at pressures between 100 MPa and 300 MPa and coincided with irreversible loss of membrane integrity as indicated by uptake of propidium iodide (PI) and leakage of protein of molecular mass between 9 and 78 kDa from the cells. Protein release increased to a maximum at 400 MPa then decreased, possibly due to intracellular aggregation at the higher pressures. In the pressure-resistant strain E. coli J1, PI was taken up during pressure treatment but not after decompression indicating that cells were able to reseal their membranes. Loss of viability in strain J1 coincided with the transient loss of membrane integrity between approximately 200 MPa and 600 MPa. In E. coli J1 leakage of protein occurred before loss of viability and the released protein was of low molecular mass, between 8 and 11 kDa and may have been of periplasmic origin. In these two strains differences in pressure resistance appeared to be related to differences in the ability of their membranes to withstand disruption by pressure. However it appears that transient loss of membrane integrity during pressure can lead to cell death irrespective of whether cells can reseal their membranes afterwards.

Influence of environmental stress on distributions of times to first division in Escherichia coli populations, as determined by digital-image analysis of individual cells.

The distributions of times to first cell division were determined for populations of Escherichia coli stationary-phase cells inoculated onto agar media. This was accomplished by using automated analysis of digital images of individual cells growing on agar and calculation of the "box area ratio." Using approximately 300 cells per experiment, the mean time to first division and standard deviation for cells grown in liquid medium at 37 degrees C and inoculated on agar and incubated at 20 degrees C were determined as 3.0 h and 0.7 h, respectively. Distributions were observed to tail toward the higher values, but no definitive model distribution was identified. Both preinoculation stress by heating cultures at 50 degrees C and postinoculation stress by growth in the presence of higher concentrations of NaCl increased mean times to first division. Both stresses also resulted in an increase in the spread of the distributions that was proportional to the mean division time, the coefficient of variation being constant at approximately 0.2 in all cases. The "relative division time," which is the time to first division for individual cells expressed in terms of the cell size doubling time, was used as measure of the "work to be done" to prepare for cell division. Relative division times were greater for heat-stressed cells than for those growing under osmotic stress.

Effect of small, acid-soluble proteins on spore resistance and germination under a combination of pressure and heat treatment.

To find the range of pressure required for effective high-pressure inactivation of bacterial spores and to investigate the role of alpha/beta-type small, acid-soluble proteins (SASP) in spores under pressure treatment, mild heat was combined with pressure (room temperature to 65 degrees C and 100 to 500 MPa) and applied to wild-type and SASP-alpha-/beta- Bacillus subtilis spores. On the one hand, more than 4 log units of wild-type spores were reduced after pressurization at 100 to 500 MPa and 65 degrees C. On the other hand, the number of surviving mutant spores decreased by 2 log units at 100 MPa and by more than 5 log units at 500 MPa. At 500 MPa and 65 degrees C, both wild-type and mutant spore survivor counts were reduced by 5 log units. Interestingly, pressures of 100, 200, and 300 MPa at 65 degrees C inactivated wild-type SASP-alpha+/beta+ spores more than mutant SASP-alpha-/beta- spores, and this was attributed to less pressure-induced germination in SASP-alpha-/beta- spores than in wild-type SASP-alpha+/beta+ spores. However, there was no difference in the pressure resistance between SASP-alpha+/beta+ and SASP-alpha-/beta- spores at 100 MPa and ambient temperature (approximately 22 degrees C) for 30 min. A combination of high pressure and high temperature is very effective for inducing spore germination, and then inactivation of the germinated spore occurs because of the heat treatment. This study showed that alpha/beta-type SASP play a role in spore inactivation by increasing spore germination under 100 to 300 MPa at high temperature.

New mathematical modeling approach for predicting microbial inactivation by high hydrostatic pressure.

A new primary model based on a thermodynamically consistent first-order kinetic approach was constructed to describe non-log-linear inactivation kinetics of pressure-treated bacteria. The model assumes a first-order process in which the specific inactivation rate changes inversely with the square root of time. The model gave reasonable fits to experimental data over six to seven orders of magnitude. It was also tested on 138 published data sets and provided good fits in about 70% of cases in which the shape of the curve followed the typical convex upward form. In the remainder of published examples, curves contained additional shoulder regions or extended tail regions. Curves with shoulders could be accommodated by including an additional time delay parameter and curves with tails shoulders could be accommodated by omitting points in the tail beyond the point at which survival levels remained more or less constant. The model parameters varied regularly with pressure, which may reflect a genuine mechanistic basis for the model. This property also allowed the calculation of (a) parameters analogous to the decimal reduction time D and z, the temperature increase needed to change the D value by a factor of 10, in thermal processing, and hence the processing conditions needed to attain a desired level of inactivation; and (b) the apparent thermodynamic volumes of activation associated with the lethal events. The hypothesis that inactivation rates changed as a function of the square root of time would be consistent with a diffusion-limited process.

A novel method for measuring lag times in division of individual bacterial cells using image analysis.

A method is presented for determining the time to first division of individual bacterial cells growing on agar media. Bacteria were inoculated onto agar-coated slides and viewed by phase-contrast microscopy. Digital images of the growing bacteria were captured at intervals and the time to first division estimated by calculating the "box area ratio". This is the area of the smallest rectangle that can be drawn around an object, divided by the area of the object itself. The box area ratios of cells were found to increase suddenly during growth at a time that correlated with cell division as estimated by visual inspection of the digital images. This was caused by a change in the orientation of the two daughter cells that occurred when sufficient flexibility arose at their point of attachment. This method was used successfully to generate lag time distributions for populations of Escherichia coli, Listeria monocytogenes and Pseudomonas aeruginosa, but did not work with the coccoid organism Staphylococcus aureus. This method provides an objective measure of the time to first cell division, whilst automation of the data processing allows a large number of cells to be examined per experiment.

Morphological and physiological changes induced by high hydrostatic pressure in exponential- and stationary-phase cells of Escherichia coli: relationship with cell death.

The relationship between a loss of viability and several morphological and physiological changes was examined with Escherichia coli strain J1 subjected to high-pressure treatment. The pressure resistance of stationary-phase cells was much higher than that of exponential-phase cells, but in both types of cell, aggregation of cytoplasmic proteins and condensation of the nucleoid occurred after treatment at 200 MPa for 8 min. Although gross changes were detected in these cellular structures, they were not related to cell death, at least for stationary-phase cells. In addition to these events, exponential-phase cells showed changes in their cell envelopes that were not seen for stationary-phase cells, namely physical perturbations of the cell envelope structure, a loss of osmotic responsiveness, and a loss of protein and RNA to the extracellular medium. Based on these observations, we propose that exponential-phase cells are inactivated under high pressure by irreversible damage to the cell membrane. In contrast, stationary-phase cells have a cytoplasmic membrane that is robust enough to withstand pressurization up to very intense treatments. The retention of an intact membrane appears to allow the stationary-phase cell to repair gross changes in other cellular structures and to remain viable at pressures that are lethal to exponential-phase cells.

The effects of hydrostatic pressure on ribosome conformation in Escherichia coli: and in vivo study using differential scanning calorimetry.

Differential scanning calorimetry of whole Escherichia coil cells allowed the detection in vivo of changes in ribosome conformation. This enabled for the first time an analysis of the effects of high hydrostatic pressures on ribosomes in living cells. A correlation was observed between loss of cell viability and decrease in ribosome-associated enthalpy in cells subjected to pressures of 50-250 MPa for 20 min. Cell death and ribosome damage were therefore closely related phenomena. In pressure-treated cells, the thermogram peak temperatures decreased, suggesting that the remaining ribosomes had adopted a less stable conformation. During subsequent incubation of the cultures at 37 degrees C, peak temperatures and enthalpies gradually increased over a period of 5 h. This change in ribosome conformation had no apparent effect on cell survival, as viability continued to decrease. The addition of 5 mM MgCl2 before pressure treatment of cells prevented the reduction in stability of surviving ribosomes but had no effect on the initial loss of enthalpy or on cell viability.