The effect of acid shock on the heat resistance of Listeria monocytogenes

The effect of acid shock on the heat resistance of Listeria monocytogenes was investigated. After growth for 24 h at 30°C in tryptic soy broth containing 0.6% yeast extract, cell culture suspensions of L. monocytogenes were acidified with HCl or acetic acid over various time periods before being heated in whole milk to a temperature of 58°C. When cells were acid-shocked immediately with HCl for 1, 2 or 4 h, those acid-shocked for 1 h demonstrated the largest increase in thermotolerance as compared to control cells, when heated at 58°C in whole milk. In fact, cells acid-shocked for longer than 1 h with HCl demonstrated in some instances a decreased recovery as compared to control cells. Other types of acid-shock treatments included lowering the pH gradually either over a 4 h or a 24 h period. However, regardless of the type of acid-shock treatment, cells acid-shocked with HCl (but not acetic acid) prior to heating had significantly greater heat resistance as compared to control (non-acid-shocked) cells. It appears that acidification with HCl prior to final heating can enhance the heat resistance of L. monocytogenes.     

Physiological and biochemical studies on psychrotolerance in Listeria monocytogenes

Studies were undertaken to explain the ability of Listeria monocytogenes to grow at low temperatures in a chemostat. It was found that when grown in continuous culture at a dilution rate of 0·02 h⁻¹ L. monocytogenes had a lower proportion of anteiso -17:0, and a higher proportion of anteiso -15:0, and smaller chain fatty acids when grown at 10°C compared to 30°C. A previously unreported glycolipid was only seen after growth at low temperature. Growth temperature had no effect on the rate of glucose uptake.     

Effect of culture age, pre-incubation at low temperature and pH on the thermal resistance of Aeromonas hydrophila

S. CONDON, M.L. GARCIA, A. OTERO AND F.J. SALA. 1992. The thermal resistance of Aeromonas hydrophila strain NCTC 8049 was determined within the range 48°-65°C with a thermoresistometer TR-SC and McIlvaine buffer. The effects of culture age, pre-incubation at 7°C and the pH of the heating menstruum were evaluated. The pattern of thermal death was dependent on culture age. Cells heated in the late logarithmic growth phase (15 h at 30°C) were twice as resistant as those in the early stage (5 h at 30°C), and the maximum D -value was obtained after 72 h incubation (5.5 total increase). The age of the cells did not affect z -values significantly. The heat resistance of cells incubated for 48 h at 30°C increased (twice) after holding at 7°C for 72 h. Pre-incubation at low temperature of older cultures (72 h, 30°C) did not influence their D -values. Maximum heat resistance was found at pH 6.0 and minimal at pH 4.0. Decreasing the pH from 6.0 4.0 reduced D -values by a factor of 5. Although the strain studied was heat-sensitive ( D _55°C= 0.17 min; z = 5.11°C), survivor curves of cultures older than 50 h showed a significant tailing. Organisms surviving in the tails were only slightly more resistant than were the original population.     

Resistance of Escherichia coli grown at different temperatures to various environmental stresses

Aims:  To study the influence of growth temperature on the resistance of Escherichia coli to three agents of different nature: heat, pulsed electric field (PEF) and hydrogen peroxide.
Methods and Results:  Escherichia coli cells were grown to stationary phase at 10°C, 20°C, 30°C, 37°C and 42°C. Survival curves to a heat treatment at 57·5°C, to a PEF treatment at 22 kV cm⁻¹ and to 40 mmol l⁻¹ hydrogen peroxide were obtained and fitted to a model based on the Weibull distribution to describe and compare the inactivation. Time to inactivate the first log cycle of the population at 57·5°C of cells grown at 42°C was sixfold higher than that corresponding to cells grown at 10°C. On the contrary, cells grown at 10°C and 20°C were more resistant to PEF and hydrogen peroxide treatments.
Conclusions:  The influence of growth temperature on bacterial resistance depends on the stress applied. Cells grown at higher temperatures were more heat resistant, but more sensitive to PEF and hydrogen peroxide.
Significance and Impact of the Study:  Results obtained in this investigation help in understanding the physiology of bacterial resistance and the inactivation mechanisms of different technologies.     

Heat resistance of Listeria monocytogenes in dairy products as affected by the growth medium

Listeria monocytogenes strains 1151 and Scott A were grown in broth at 30 °C and transferred to half cream, double cream and butter stored at 5 °C to determine the influence of dairy product composition on heat resistance at 52, 56, 60, 64 and 68 °C. Strain 1151 showed a higher heat resistance than strain Scott A. The heat resistance of both strains was higher in the dairy products than in broth, particularly at lower temperatures. A significant difference was observed between log 10 of the D -values in the different dairy products. The D -values obtained for both strains resuspended in all the dairy products would result in efficient elimination of the pathogen at 72·7 °C for 15 s. The highest D -value was 11·30 s at 68 °C and by using a z -value of 6·71 °C it can be determined that at 72·7 °C the D -value would be 1·5 s. The 15 s process would therefore achieve 10 log reductions. The effect of growth conditions on the heat resistance at 60 °C of L. monocytogenes Scott A was also investigated. When the cells were grown in the dairy products themselves, and particularly butter, the heat resistance of Scott A was enhanced; for example, the D -values were 7·15 times higher than in broth. Further studies are required to investigate if this protection against heating exists at higher temperatures, in which case the efficiency of pasteurization treatments or other heat treatments would be considerably lowered.     

Effect of pre- and post-heat shock temperature on the persistence of thermotolerance and heat shock-induced proteins in Listeria monocytogenes

The effect of incubation temperature, before and after a heat shock, on thermotolerance of Listeria monocytogenes at 58°C was investigated. Exposing cells grown at 10°C and 30°C to a heat shock resulted in similar rises in thermotolerance while the increase was significantly higher when cells were grown at 4°C prior to the heat shock. Cells held at 4°C and 10°C after heat shock maintained heat shock-induced thermotolerance for longer than cells held at 30°C. The growth temperature prior to inactivation had negligible effect on the persistence of heat shock-induced thermotolerance. Concurrent with measurements of thermotolerance were measurements of the levels of heat shock-induced proteins. Major proteins showing increased synthesis upon the heat shock had approximate molecular weights of 84, 74, 63, 25 and 19 kDa. There was little correlation between the loss of thermotolerance after the heat shock and the levels of these proteins. Thermotolerance of heat shocked and non-heat shocked cells was described by traditional log-linear kinetics and a model describing a sigmoidal death curve (logistic model). Employing log-linear kinetics resulted in a poor fit to a major part of the data whereas a good fit was achieved by the use of a logistic model.     

Significance of temperature and preincubation temperature on survival of Listeriamonocytogenes at pH 4·8

Listeria monocytogenes is a food-borne pathogenic bacterium that can be found in softcheese. At the beginning of cheese ripening, the pH is about 4·85–4·90. The aimof this work was to study the influence of temperature, preincubation temperature (temperature atwhich the inoculum was cultivated) and initial bacterial concentration on the survival of L.monocytogenes (strain Scott A) at pH 4·8. It was demonstrated in an earlier study thatthese factors did influence growth kinetics. Survival studies of L. monocytogenes weredone in a laboratory broth simulating cheese composition. Four test temperatures (2, 6, 10 and14°C) and two preincubation temperatures were studied (30°C or the test temperature). Listeria monocytogenes (strain Scott A) was unable to grow at pH 4·8 under allconditions tested. The time for 10% survival was about 11 and 2 d, at 2°C with preincubationat 2°C and 30°C, respectively; 9 d at 6°C with preincubation at 6°C; 4 d at 6°Cwith preincubation at 30°C; and 1 d at 14°C with preincubation at 14°C or at 30°C.The results show that survival of L. monocytogenes (strain Scott A) at pH 4·8 is notdependent on initial bacterial concentration but on both the test and preincubation temperatures.     

The effect of various acidulants on the growth of Listeria monocytogenes

The ability of four Listeria monocytogenes strains to initiate growth in brain heart infusion broth adjusted to various pH values with either acetic, lactic, citric or hydrochloric acid was investigated. Acetic acid was the most effective inhibitor tested, since in broth adjusted with this acid a higher minimum pH was required for growth of the various strains at both 4 and 30°C, as compared with broth adjusted with the other acidulants. The minimum pH value required for the initiation of growth of L. monocytogenes ranged from 5·0 to 5·7 at 4°C, and from 4·3 to 5·2 at 30°C, depending upon the acidulant used.     

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 degrees 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.     

Modelling the effect of the redox potential and pH of heating media on Listeria monocytogenes heat resistance

Aims:  Study the effect of redox potential and pH of the heating media on Listeria monocytogenes heat resistance and model its action at fixed temperature.
Methods and Results:  The heat resistance of Listeria monocytogenes at 58°C was studied in Brain Heart Infusion broth as a function of pH (from 5·0 to 7·0) and redox potential ( E _h7). The media redox was adjusted with nitrogen gas, potassium ferricyanide and dithiothreitol. A Weibull model was used to fit survival curves. The heat resistance parameter (δ_58°C) was estimated from each inactivation curve. A major effect of pH was observed. Bigelow model was used to describe the effect of redox potential on the apparent L. monocytogenes heat resistance. The highest δ_58°C values have been obtained at pH 7·0 and oxidizing conditions.
Conclusions:  The developed model indicates that the E _h7 has a significant effect and varied depending on the pH of the heating media. The z _redox values, calculated from δ_58°C allowed quantifying the influence of heating media redox potential on L. monocytogenes thermal inactivation.
Significance and Impact of the Study:  The obtained model shows the action of redox potential on L. monocytogenes thermal destruction and might be useful to take into account in food thermal processes.     

Environmental factors influencing the inactivation of Listeria monocytogenes by pulsed electric fields

AIMS: To investigate the influence of the growth phase, growth temperature, storage time, pH and aw of the treatment medium on the resistance of Listeria monocytogenes to pulsed electric fields (PEF). METHODS AND RESULTS: Square wave pulses of 2 micros at a frequency of 1 Hz and 25 and 28 kV cm(-1) were used. Cells were more PEF resistant in the stationary than in the exponential phase at both incubation temperatures investigated (4 and 35 degrees C). Cells grown at 4 degrees C were more PEF sensitive than cells grown at 35 degrees C independent of the growth phase. After a treatment of 25 kV cm(-1) and 800 micros, 1.48, 3.86 and 5.09 log10 cycles of inactivation were obtained at pH 7.0, 5.4 and 3.8, respectively. A reduction in the aw of the treatment medium protected cells against PEF treatments. CONCLUSIONS: The PEF resistance of L. monocytogenes depended on different environmental factors. The influence of growth conditions and treatment medium characteristics should be known and controlled to obtain reproducible and reliable PEF inactivation data. SIGNIFICANCE AND IMPACT OF THE STUDY: Erroneous conclusions and misinterpretation of results are possible if factors affecting the PEF resistance of L. monocytogenes are not considered during PEF inactivation studies.     

Effect of growth conditions on heat resistance of Arizona bacteria grown in a chemostat.

The effects of various growth conditions on the heat resistance of Arizona bacteria grown in a continuous-culture device (chemostat) were studied. Using either glucose, NH4Cl, NaH2PO4, or MgCl2 as the rate-limiting nutrient, it was found that the heat resistance, in all cases depended on the dilution rate and, hence, growth rate of the culture. Cells grown at high dilution rates were less heat resistant than those grown at low dilution rates. If, however, the dilution rate was maintained at a constant rate, the higher the growth temperature, the more heat resistant were the cells. Also at any given dilution rate, the cells were most heat resistant when grown at a near neutral pH. Most survival curves were biphasic in shape, indicating the presence in the population of two fractions of cells, one fraction being more resistant than the other. The size of the more heat-resistant fraction varied from almost 100% in very slow-growing cultures to practically 0% in cultures grown at a dilution rate of 0.67 h-1.     

Effects of gaseous environment and temperature on the storage behaviour of Listeria monocytogenes on chicken breast meat

Portions of skinless chicken breast meat (pH 5·8) were inoculated with a strain of Listeria monocytogenes and stored at 1, 6 or 15°C in (1) aerobic conditions; (2) 30% CO₂+ air; (3) 30% CO₂+ N₂; and (4) 100% CO₂. When samples were held at 1°C the organism failed to grow under any of the test conditions, despite marked differences between treatments in spoilage rate and ultimate microflora. At 6°C counts of L. monocytogenes increased ca 10-fold in aerobic conditions before spoilage of the meat, but only when the inoculum culture was incubated at 1°C rather than 37°C. In CO₂ atmospheres growth of L. monocytogenes was inhibited on meat held at 6°C, especially under 100% CO₂. By contrast, storage at 15°C led to spoilage of the meat within 2 d, in all gaseous environments, and listeria levels increased up to 100-fold. Differences in the behaviour of L. monocytogenes on poultry and red meats are discussed.     

Nisin induces changes in membrane fatty acid composition of Listeria monocytogenes nisin-resistant strains at 10°C and 30°C

Listeria monocytogenes isolates resistant to 10⁵ IU ml^-1 nisin were obtained at 30°C (NR30) and at 10°C (NR10). Nisin prolonged the lag phase of isolate NR30 at 10°C. Isolates NR30 and NR10 did not produce a nisinase. Protoplasts of isolate NR30 were unaffected by exposure to nisin. The fatty acid composition from the wild-type strain and NR isolates was determined. As expected, temperature-induced differences in the C15/C17 fatty acid ratios were found. Growth of the NR strains in the presence of nisin resulted in significantly different C15/C17 ratios and a significant increase in the percentage of C16:0, C16: 1, C18:0 and C18: 1 fatty acids at 10°C and 30°C. Both the NR10 and NR30 isolates had similar growth rates at low temperatures, but these were slower than the wild-type strain. These results indicate that 'nisin resistance'is an environmentally defined phenotype and that nisin induces changes in the fatty acid composition of the membrane in L. monocytogenes nisin-resistant isolates regardless of the growth temperature.     

Expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments

AIMS: To study the expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies (MAbs) C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments. METHODS AND RESULTS: The reaction patterns of antibodies to L. monocytogenes held in stressful environments for a short duration (3 h) or grown for extended periods (16-72 h) were investigated. During both short or prolonged exposure to stress environments of high temperature (45 degrees C) and NaCl (>1.5%, w/v), reactions of whole cells of L. monocytogenes to antibodies were severely affected as determined by ELISA and by the reduced expression of the antibody-reactive 66 kDa antigen in the Western blot assay. Conversely, cold (4-15 degrees C) or acid (pH 2-3) stress environments had very little effect on antigen expression or antibody reaction. Additionally, heat-killed cells showed reduced reactions to these antibodies when compared with unheated cells. Artificially created stress environments in hotdog slurry also affected the antigen expression in L. monocytogenes. Immunoelectron microscopy revealed that the antibody-reactive antigens were uniformly present on the surface of the cells. Morphological characteristics following growth in stressed environments revealed that heat stress at 45 degrees C caused L. monocytogenes cells to be elongated and to form clumps; whereas, osmotic stress (5.5% NaCl, w/v) caused filamentous appearance with multiple septa along the length of the cell. CONCLUSIONS: These results indicated that MAb C11E9 or EM-7G1 could detect L. monocytogenes from cold or acid-stress environments; however, they may show weaker reactions with heat or osmotically stressed cells or cells grown at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteria in food are routinely subjected to various stresses, induced by cold, heat, salt or acid during processing and storage. Whether stresses would modify the expression of cellular antigens of L. monocytogenes is of a great concern for immunodetections in food products.     

Factors controlling acid tolerance of Listeria monocytogenes: effects of nisin and other ionophores.

The acid tolerance of a Listeria monocytogenes serotype 4b strain was studied by measuring its ability to survive at an acidic pH at 37 degrees C. The acid tolerance of L. monocytogenes was much lower than those of Escherichia coli O157:H7 and Shigella flexneri strains. This observation suggested a higher infective dose for L. monocytogenes than E. coli O157:H7 and Shigella. The susceptibility of L. monocytogenes to acidic pH was dependent upon growth medium pH and growth phase of the culture. Nisin and some other ionophores reduced the acid tolerance of both stationary-phase and log-phase cultures of L. monocytogenes. These studies indicated that nisin might be a useful candidate for controlling acid tolerance of L. monocytogenes.     

A comparison of low temperature growth vs low temperature shifts to induce resistance to photoinhibition in spinach (Spinacia oleracea)

Plants of Spinacia oleracea L. cv. Savoy grown under cold-hardening (5°C) and nonhardening (16°C) conditions were exposed to a photoinhibitory irradiance of 1300 μmol rrr^: m-² S-¹ 5°C for 12 h. Plants grown at 5°C exhibited a greater resistance to photoinhibition at low temperature in comparison to plants grown at 16°C as measured by the photochemical efficiency of photosyslem II. In contrast, tuily expanded leaves of plants grown at 16°C and then shifted to 5°C for 10 days did not exhibit increased resistance to photoinhibition. This was observed irrespective of the phoioperiod experienced during the shift to a lower temperature. Furthermore, spinach grown at 16°C and subsequently exposed to a stepped, daily decrease in temperature from 16 to 1°C over 10 days w ith a concomitant reduction in photoperiod. also did not exhibit any change in susceptibility to photoinhibition. Thus, a decrease in photoperiod accompanied by either an abrupt or stepped low temperature shift cannot induce increased resistance to photoinhibition. This confirms the hypothesis that growth and development at cold-hardening temperature are absolute requirements for the acquisition of resistance to photoinhibition at low temperature.     

Expression of superoxide dismutase in Listeria monocytogenes.

The nature and expression of superoxide dismutase (SOD; EC 1.15.1.1) in the gram-positive food-borne pathogen Listeria monocytogenes were examined. Metal depletion and reconstitution studies and resistance to H2O2 and potassium cyanide inactivation indicated that L. monocytogenes has a single SOD which utilizes manganese as a metal cofactor. The specific activity of SOD was unchanged in cells exposed to a heat shock at 42 degrees C or grown in the presence of paraquat-generated superoxide anion or of metal chelators in the medium. SOD levels increased, however, as the cells progressed through the logarithmic phase of growth and into the stationary phase. Furthermore, SOD activity decreased with decreasing growth temperatures and declined concurrently with decreased growth when higher concentrations of sodium chloride were added to the medium. Cells grown anaerobically possessed relatively high levels of SOD, although these levels were about 10 to 30% lower than those of aerobically grown bacteria. Different isolates of L. monocytogenes were found to produce approximately equivalent levels of SOD, although greater differences in SOD expression were seen among other species of Listeria. When compared with L. monocytogenes, for example, Listeria welshimeri typically produced about 30% greater SOD activity, whereas Listeria murrayi produced about 60% less total SOD activity. Although all species of Listeria produced a single Mn-type SOD, differences in the relative electrophoretic mobility of the native enzymes were noted. These data suggest that the single L. monocytogenes SOD enzyme is constitutively produced in response to many environmental factors and may also be responsive to the cellular growth rate.     

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).