Combined effect of sodium nitrite with high-pressure treatments on the inactivation of Escherichia coli BW25113 and Listeria monocytogenes NCTC 11994.

The bactericidal effect of acidified sodium nitrite alone or when combined with high hydrostatic pressure (HHP) treatment was examined with Escherichia coli BW25113 and Listeria monocytogenes NCTC 11994. A powerful synergistic effect of HHP plus nitrite was observed at pH 4·0, but not at higher pH values. Escherichia coli hmpA and norV mutants lacking defences against nitrosative stress were more sensitive to pressure combined with acidified sodium nitrite than the wild-type strain, suggesting an involvement of nitric oxide in the bactericidal effect.

Environmental factors influencing the inactivation of Cronobacter sakazakii by high hydrostatic pressure.

The effect of High Hydrostatic Pressure (HHP) on the survival of Cronobacter sakazakii was investigated. Deviations from linearity were found on the survival curves and the Mafart equation accurately described the kinetics of inactivation. Comparisons between strains and treatments were made based on the time needed for a 5-log(10) reduction in viable count. The ability of C. sakazakii to tolerate high pressure was strain-dependent with a 26-fold difference in resistance among four strains tested. Pressure resistance was greatest in the stationary growth phase and at the highest growth temperatures tested (30 and 37 °C). Cells treated in neutral pH buffer were 5-fold more resistant than those treated at pH 4.0, and 8-fold more sensitive than those treated in buffer with sucrose added (a(w)=0.98). Pressure resistance data obtained in buffer at the appropriate pH adequately estimated the resistance of C. sakazakii in chicken and vegetables soups. In contrast, a significant protective effect against high pressure was conferred by rehydrated powdered milk. As expected, treatment efficacy improved as pressure increased. z values of 112, 136 and 156 MPa were obtained for pH 4.0, pH 7.0 and a(w)=0.98 buffers, respectively. Cells with sublethal injury to their outer and cytoplasmic membranes were detected after HHP under all the conditions tested. The lower resistance of C. sakazakii cells when treated in media of pH 4.0 seemed to be due to a decreased barostability of the bacterial envelopes. Conversely, the higher resistance displayed in media of reduced water activity may relate to a higher stability of bacterial envelopes.

Modeling the variability of single-cell lag times for Listeria innocua populations after sublethal and lethal heat treatments.

Optical density measurements were used to estimate the effect of heat treatments on the single-cell lag times of Listeria innocua fitted to a shifted gamma distribution. The single-cell lag time was subdivided into repair time (the shift of the distribution assumed to be uniform for all cells) and adjustment time (varying randomly from cell to cell). After heat treatments in which all of the cells recovered (sublethal), the repair time and the mean and the variance of the single-cell adjustment time increased with the severity of the treatment. When the heat treatments resulted in a loss of viability (lethal), the repair time of the survivors increased with the decimal reduction of the cell numbers independently of the temperature, while the mean and variance of the single-cell adjustment times remained the same irrespective of the heat treatment. Based on these observations and modeling of the effect of time and temperature of the heat treatment, we propose that the severity of a heat treatment can be characterized by the repair time of the cells whether the heat treatment is lethal or not, an extension of the F value concept for sublethal heat treatments. In addition, the repair time could be interpreted as the extent or degree of injury with a multiple-hit lethality model. Another implication of these results is that the distribution of the time for cells to reach unacceptable numbers in food is not affected by the time-temperature combination resulting in a given decimal reduction.

Predicting the thermal inactivation of bacteria in a solid matrix: simulation studies on the relative effects of microbial thermal resistance parameters and process conditions.

A combined mathematical model for predicting heat penetration and microbial inactivation in a solid body heated by conduction was tested experimentally by inoculating agar cylinders with Salmonella typhimurium or Enterococcus faecium and heating in a water bath. Regions of growth where bacteria had survived after heating were measured by image analysis and compared with model predictions. Visualisation of the regions of growth was improved by incorporating chromogenic metabolic indicators into the agar. Preliminary tests established that the model performed satisfactorily with both test organisms and with cylinders of different diameter. The model was then used in simulation studies in which the parameters D, z, inoculum size, cylinder diameter and heating temperature were systematically varied. These simulations showed that the biological variables D, z and inoculum size had a relatively small effect on the time needed to eliminate bacteria at the cylinder axis in comparison with the physical variables heating temperature and cylinder diameter, which had a much greater relative effect.

Factors affecting the pressure resistance of some Campylobacter species.

AIMS: To compare pressure resistance between strains of Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus, and to investigate the effect of suspending medium on pressure resistance of sensitive and more resistant strains. METHODS AND RESULTS: Six strains of C. jejuni and four each of C. coli, C. lari and C. fetus were pressure treated for 10 min at 200 and 300 MPa. Individual strains varied widely in pressure resistance but there were no significant differences between the species C. jejuni, C. coli and C. lari. Campylobacter fetus was significantly more pressure sensitive than the other three species. The pressure resistance of C. jejuni cultures reached a maximum at 16-18 h on entry into stationary phase then declined to a minimum at 75 h before increasing once more. Milk was more baroprotective than water, broth or chicken slurry but did not prevent inactivation even of a resistant strain at 400 MPa. CONCLUSIONS: Pressure resistance varies considerably between species of Campylobacter and among strains within a species, and survival after a pressure challenge will be markedly influenced by culture age and food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Despite the strain variation in pressure resistance and protective effects of food, Campylobacter sp. do not present a particular problem for pressure processing.

Physiological changes in Campylobacter jejuni on entry into stationary phase.

Campylobacter jejuni NCTC 11168 does not exhibit the general increase in cellular stress resistance on entry into stationary phase that is seen in most other bacteria. This is consistent with the lack of global stationary phase regulatory elements in this organism, deduced from an analysis of its genome sequence. We now show that C. jejuni NCTC 11168 does undergo certain changes in stationary phase, of a pattern not previously described. As cells entered stationary phase there was a change in membrane fatty acid composition, principally a decrease in the proportion of unsaturated fatty acids and an increase in the content of cyclopropane and short-chain fatty acids. These changes in membrane composition were accompanied by an increase in the resilience of the cell membrane towards loss of integrity caused by pressure and an increase in cellular pressure resistance. By contrast, there were no major changes in resistance to acid or heat treatment. A similar pattern of changes in stress resistance on entry into stationary phase was seen in C. jejuni NCTC 11351, the type strain. These changes appear to represent a restricted physiological response to the conditions existing in stationary phase cultures, in an organism having limited capacity for genetic regulation and adaptation to environment.

Emergence of variants with altered survival properties in stationary phase cultures of Campylobacter jejuni.

During the stationary phase of Campylobacter jejuni NCTC 11351 viable numbers fluctuate in a characteristic fashion. After reaching the maximum cell count (ca. 2 x 10(9) CFU/ml) in early stationary phase (denoted phase 1), viable numbers subsequently decrease to about 10(6) CFU/ml after 48 h and then increase again to about 10(8) CFU/ml (denoted phase 2) before decreasing once more to a value intermediate between the previous maximum and minimum values. To investigate whether the increase in viable numbers following the initial decline was due to the emergence of a new strain with a growth advantage in stationary phase analogous to the 'GASP' phenotype described in Escherichia coli [Science 259 (1993) 1757], we conducted mixed culture experiments with cells from the original culture and antibiotic-resistant marked organisms isolated from the re-growth phase. In many experiments of this type, strains isolated from phase 2 failed to out-compete the original strain and we have thus been unable to demonstrate a convincing GASP phenotype. However, strains isolated from phase 2 showed a much lower rate of viability loss in early stationary phase and a small increase in resistance to aeration, peroxide challenge and heat, indicating that the emergent strain was different from the parent. These results support the view that dynamic population changes occur during the stationary phase of C. jejuni that may play a role in the survival of this organism.

Description of a "phoenix" phenomenon in the growth of Campylobacter jejuni at temperatures close to the minimum for growth.

When Campylobacter jejuni cultures that had been grown in broth at 39 degrees C were subcultured into fresh medium at 30 degrees C, there was a transient period of growth followed by a decline in viable-cell numbers before growth resumed once more. We propose that this complex behavior is the net effect of the growth of inoculum cells followed by a loss of viability due to oxidative stress and the subsequent emergence of a spontaneously arising mutant population that takes over the culture.

Role of membrane fluidity in pressure resistance of Escherichia coli NCTC 8164.

The relationship among growth temperature, membrane fatty acid composition, and pressure resistance was examined in Escherichia coli NCTC 8164. The pressure resistance of exponential-phase cells was maximal in cells grown at 10 degrees C and decreased with increasing growth temperatures up to 45 degrees C. By contrast, the pressure resistance of stationary-phase cells was lowest in cells grown at 10 degrees C and increased with increasing growth temperature, reaching a maximum at 30 to 37 degrees C before decreasing at 45 degrees C. The proportion of unsaturated fatty acids in the membrane lipids decreased with increasing growth temperature in both exponential- and stationary-phase cells and correlated closely with the melting point of the phospholipids extracted from whole cells examined by differential scanning calorimetry. Therefore, in exponential-phase cells, pressure resistance increased with greater membrane fluidity, whereas in stationary-phase cells, there was apparently no simple relationship between membrane fluidity and pressure resistance. When exponential-phase or stationary-phase cells were pressure treated at different temperatures, resistance in both cell types increased with increasing temperatures of pressurization (between 10 and 30 degrees C). Based on the above observations, we propose that membrane fluidity affects the pressure resistance of exponential- and stationary-phase cells in a similar way, but it is the dominant factor in exponential-phase cells whereas in stationary-phase cells, its effects are superimposed on a separate but larger effect of the physiological stationary-phase response that is itself temperature dependent.

The effect of inoculum size and sublethal injury on the ability of Listeria monocytogenes to initiate growth under suboptimal conditions.

AIMS: To investigate the effect of inoculum size and physiological state on the ability of Listeria monocytogenes cells to initiate growth under suboptimal conditions of salt concentration and pH. METHODS AND RESULTS: Cell suspensions were serially diluted in media of different salt concentration or pH and replicate inocula distributed into 96-well microplates. The proportion of wells showing growth at each dilution level was determined after incubation for 6 weeks for each set of conditions. Growth occurred from single cells up to a concentration of 1.2 mol l-1 NaCl; above this threshold, the inoculum size needed to initiate growth became progressively larger. A similar effect was seen with decreasing pH but only very close to the growth/no growth boundary. The threshold for inoculum-dependent growth was lower in exponential phase cells than in stationary phase ones and sublethal injury greatly decreased the probability of growth from small inocula. CONCLUSIONS: The growth/no growth boundary for L. monocytogenes is not an absolute cut-off point but represents a region where the probability of growth rapidly decreases as conditions become more extreme. We interpret the requirement for a critical inoculum size for growth as being due to death of a proportion of cells in the inoculum rather than to co-operative population effects. SIGNIFICANCE AND IMPACT OF THE STUDY: Physiological heterogeneity within the cell population and inoculum size will affect the risk of L. monocytogenes growing in food.

The recovery of Arcobacter butzleri NCTC 12481 from various temperature treatments.

AIMS: The aim of this study was to determine the growth and survival characteristics for Arcobacter butzleri NCTC 12481. METHODS AND RESULTS: The temperature and pH growth ranges were 15-39 degrees C and pH 6.0-8.0, as determined using impedance microbiology. The maximum specific growth rate was 00.57 h(-1) at 30 degrees C, pH 7.0. Arcobacter butzleri harvested from the exponential phase was more resistant to heat treatment than stationary phase cells (D55 1.1 and 0.4 min, respectively). Fluorescent dye uptake, and the release of UV-absorbing material, increased in heat-treated cells. After 21 d storage at 4 and -20 degrees C, A. butzleri was recovered on blood agar, but not on the isolation media CAT or CCDA. CONCLUSION: Arcobacter butzleri cells from the exponential phase were less heat sensitive than those from the stationary phase. The organism was able to survive cold storage for at least 3 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: The growth and survival characteristics have been quantified thus providing a greater understanding of this newly emerging pathogen.

Variation in resistance to high hydrostatic pressure and rpoS heterogeneity in natural isolates of Escherichia coli O157:H7.

Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.

Inactivation and injury of pressure-resistant strains of Escherichia coli O157 and Listeria monocytogenes in fruit juices.

AIMS: To investigate methods for inactivating a pressure-resistant strain of Escherichia coli O157 in fruit juices. METHODS AND RESULTS: Cells of a pressure-resistant strain of E. coli O157 (C9490) were exposed to pressures of between, 0.1 and 500 MPa for 5 min in orange, apple or tomato juice. Treatment at 500 MPa achieved an immediate reduction of 5 log units in apple juice (pH 3.5) and tomato juice (pH 4.1), but only about a 1-2 log10 reduction in orange juice (pH 3.8). The greater level of inactivation in tomato juice than in orange juice of lower pH was due to the presence of low levels (0.7%) of salt in the tomato juice. With the type-strain of E. coli (ATCC 11775) and Listeria monocytogenes NCTC 11994, similar levels of inactivation were achieved at pressures 200 MPa lower. Following storage of pressure-treated orange juice at 4 degrees C for 24 h or 25 degrees C for 3 h, the level of inactivation of E. coli O157 strain C9490 increased to 4.4 or > 7 log10 units, respectively. CONCLUSION: Treatment at 500 MPa may be insufficient to achieve a '5D' reduction in counts of pressure-resistant strains of E. coli, but subsequent death during storage substantially increases process lethality. SIGNIFICANCE AND IMPACT OF THE STUDY: Commercially-practicable pressure processes can be used to inactivate even the most pressure-and acid-resistant strains of E. coli O157, provided that processing and subsequent storage conditions are carefully optimized.

Survival of Campylobacter jejuni during stationary phase: evidence for the absence of a phenotypic stationary-phase response.

When Campylobacter jejuni NCTC 11351 was grown microaerobically in rich medium at 39 degrees C, entry into stationary phase was followed by a rapid decline in viable numbers to leave a residual population of 1% of the maximum number or less. Loss of viability was preceded by sublethal injury, which was seen as a loss of the ability to grow on media containing 0.1% sodium deoxycholate or 1% sodium chloride. Resistance of cells to mild heat stress (50 degrees C) or aeration was greatest in exponential phase and declined during early stationary phase. These results show that C. jejuni does not mount the normal phenotypic stationary-phase response which results in enhanced stress resistance. This conclusion is consistent with the absence of rpoS homologues in the recently reported genome sequence of this species and their probable absence from strain NCTC 11351. During prolonged incubation of C. jejuni NCTC 11351 in stationary phase, an unusual pattern of decreasing and increasing heat resistance was observed that coincided with fluctuations in the viable count. During stationary phase of Campylobacter coli UA585, nonmotile variants and those with impaired ability to form coccoid cells were isolated at high frequency. Taken together, these observations suggest that stationary-phase cultures of campylobacters are dynamic populations and that this may be a strategy to promote survival in at least some strains. Investigation of two spontaneously arising variants (NM3 and SC4) of C. coli UA585 showed that a reduced ability to form coccoid cells did not affect survival under nongrowth conditions.

The effect of inoculum size on the lag phase of Listeria monocytogenes.

The effect of inoculum size on population lag times of Listeria monocytogenes was investigated using the Bioscreen automated microtitre plate incubator and reader. Under optimum conditions, lag times were little affected by inoculum size and there was little variation between replicate inocula even at very low cell numbers. However, in media containing inhibitory concentrations of NaCl, both the mean lag time and variation between replicate inocula increased as the inoculum size became smaller. The variation in lag time of cells within a population was investigated in more detail by measuring the distribution of detection times from 64 replicate inocula containing only one or two cells capable of initiating growth. The variance of the lag time distribution increased with increasing salt concentration and was greater in exponential than in stationary phase inocula. The number of cells required to initiate growth increased from one cell under optimum conditions to 10(5) cells in medium with 1.8 M NaCl. The addition of spent medium from a stationary phase culture reduced the variance and decreased lag times. The ability to initiate growth under severe salt stress appears to depend on the presence of a resistant sub-fraction of the population, although high cell densities assist adaptation of those resistant cells to the unfavourable growth conditions by some unspecified medium conditioning effect. These results are relevant to the prediction of lag times and probability of growth from low numbers of stressed cells in food.

Biosynthetic requirements for the repair of membrane damage in pressure-treated Escherichia coli.

Cells of Escherichia coli that survived pressure treatment at 400 MPa showed increased sensitivity to sodium deoxycholate or sodium chloride in the plating medium, implying that homeostatic or barrier functions associated with outer and cytoplasmic membranes, respectively, were impaired. Repair of such sublethal membrane damage occurred when cells were incubated at 37 degrees C in tryptone soya broth. Inhibitor studies indicated that repair of cytoplasmic membrane damage was energy-dependent and required RNA and protein synthesis, whereas repair of outer membrane damage occurred with no requirement for energy or RNA or protein synthesis.

Detection of Salmonella enteritidis by reverse transcription-polymerase chain reaction (PCR).

A reverse transcription-polymerase chain reaction (RT-PCR) method was developed for detecting mRNA from the sefA gene of Salmonella enteritidis. Detection of target mRNA was examined from cells grown in buffered peptone water at different temperatures (37, 25 and 15 degrees C) and pH (5.5, 7.2 and 8.5). The results revealed that the levels of transcription of the sefA gene differed depending upon the physiological state of the cells. This affected the sensitivity of the RT-PCR assay. When the assay was evaluated for the detection of S. enteritidis PT4 in artificially contaminated minced beef and whole egg samples, an enrichment step was used (buffered peptone water, pH 7.2, 37 degrees C, 16 h) to increase the sensitivity of the assay. In the presence of the normal background flora of each food type, it was possible to detect ten cells of S. enteritidis PT4 after a 16-h enrichment using the RT-PCR assay, with a total testing time of 28 h. Unlike the PCR test for the sefA gene that was tested in parallel, the RT-PCR assay did not detect nonviable (heat-inactivated) S. enteritidis PT4 cells. The results supported the usefulness of RT-PCR as a method for the detection of viable microorganisms.

Visualization and modelling of the thermal inactivation of bacteria in a model food.

A large number of incidents of food poisoning have been linked to undercooked meat products. The use of mathematical modelling to describe heat transfer within foods, combined with data describing bacterial thermal inactivation, may prove useful in developing safer food products while minimizing thermal overprocessing. To examine this approach, cylindrical agar blocks containing immobilized bacteria (Salmonella typhimurium and Brochothrix thermosphacta) were used as a model system in this study. The agar cylinders were subjected to external conduction heating by immersion in a water bath. They were then incubated, sliced open, and examined by image analysis techniques for regions of no bacterial growth. A finite-difference scheme was used to model thermal conduction and the consequent bacterial inactivation. Bacterial inactivation rates were modelled with values for the time required to reduce bacterial number by 90% (D) and the temperature increase required to reduce D by 90% taken from the literature. Model simulation results agreed well with experimental results for both bacteria, demonstrating the utility of the technique.

Effect of post-treatment holding conditions on detection of tufA mRNA in ethanol-treated Escherichia coli: implications for RT-PCR-based indirect viability tests.

The PCR is a rapid and sensitive method for detecting and identifying low numbers of bacteria, but it does not discriminate between living and dead cells. Most messenger RNA (mRNA) molecules have a short half-life in the bacterial cell and their presence may therefore indicate viability. We have compared PCR and RT-PCR (targeted at tufA DNA or mRNA, respectively) for the detection of Escherichia coli, using healthy cells and those killed by exposure to different stress treatments. PCR gave a positive signal in live cells and those killed by autoclaving, boiling, or treatment with 50% ethanol, but was negative after exposure to pH 2.0 for 5 min. RT-PCR was positive in live cells but negative after all treatments except exposure to ethanol. The persistence of tufA mRNA was examined in ethanol-killed cells incubated in LB broth at different temperatures. The RT-PCR signal persisted for up to 16 h at 15 degrees C or 4 degrees C but disappeared within 2 h at 37 degrees C. RT-PCR thus has potential as an indicator of viability provided samples are pre-incubated under appropriate conditions that will ensure decay of any residual mRNA in dead cells.

The effect of the growth environment on the lag phase of Listeria monocytogenes.

The duration of lag in Listeria monocytogenes was examined in relation to the physico-chemical properties of the growth environment. It was supposed that lag would be determined by two hypothetical quantities, the amount of work that a cell has to perform to adapt to new conditions and the rate at which it can perform that work. If the rate at which the cell can perform the necessary work is a function of the maximum specific growth rate in the new environment, the hypothesis predicts that lag time should be related in some way to growth rate, provided cells are initially in approximately the same physiological state. Literature data suggest this is true for many organisms when temperature is the sole growth limiting factor. However, lag times of L. monocytogenes displayed an unusual response to temperature in which lag times of cells precultured at 37 degrees C were shorter at 15 degrees C than at 20 degrees C or 25 degrees C. Analysis of data from the Food Micromodel in which growth of L. monocytogenes was controlled by combinations of pH, NaCl concentration and temperature, showed that there was a linear relationship between lag time and mean generation time although there was much scatter in the data. When the effects of pH, solute type and concentration were investigated individually in this work the correlation between lag time and mean generation time was often poor. It would thus appear that the relationship between growth environment and lag time is more complex than the corresponding relationship between growth environment and maximum specific growth rate.