Minimum growth temperatures of Listeria monocytogenes and non-haemolytic Listeria

Minimum growth temperatures and those of decreased growth were determined for 100 strains of listerias. The ability of 78 strains of Listeria monocytogenes isolated from animals and 22 non-haemolytic strains to grow at low temperatures was studied, using a flooding technique, in a plate-type continuous temperature gradient incubator at temperatures between -1.6 and 14.5 degrees C. The mean minimum temperature for L. monocytogenes was +1.7 +/- 0.5 degrees C. The growth of non-haemolytic listerias was unobservable at +1.7 +/- 0.5 degrees C. The L. monocytogenes strains grew at about 0.6 degrees C lower than the non-pathogenic strains. No differences in growth temperatures were observed among L. monocytogenes strains isolated from different sources. The serovars with the OI antigen grew at lower temperatures (+1.0 +/- 0.3 degrees C) than the other common serovar 4b (+1.3 +/- 0.4 degrees C). The results indicate that L. monocytogenes grows better than non-haemolytic strains under cold conditions. The possible role of haemolysins as growth factors is also discussed.     

Survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer

The survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer was investigated. Raw milk was inoculated with different initial concentrations of L. monocytogenes and heated at temperatures ranging from 69° to 73°C. Listerias were not isolated from any of the milk samples immediately after thermal treatment. They were isolated, however, from 46.6% of heated samples (none from samples heated at 73°C) after variable periods at refrigeration temperature. The results suggest that a low number of listerias survive some thermal treatments, but a cold enrichment is necessary to repair the thermally injured cells and detect these organisms in milk. The importance of the isolation technique in the recovery of listerias from pasteurized milk samples is discussed.     

Thermal inactivation of Listeria monocytogenes during a process simulating temperatures achieved during microwave heating

Conventional heating was used to expose cells of Listeria monocytogenes , either in broth or in situ on chicken skin, to the mean times and temperatures that are achieved during a 28 min period of microwave cooking of a whole chicken. Heating L. monocytogenes by this method in culture broth resulted in a reduction in viable cell numbers by a factor of greater than 10⁶ upon reaching 70°C. Simulated microwave cooking of L. monocytogenes in situ , on chicken skin, resulted in more variability in the numbers of survivors. Heating for the full cook time of 28 min, however, resulted in a mean measured temperature of 85°C and no surviving listerias were detected. This indicated a reduction in viable numbers of greater than 10⁶. To reduce temperature variation, cells were heated on skin in a submerged system in which exposure to 70°C for 2 min resulted in a reduction in viable cell numbers of all strains of listerias tested of between 10⁶ and 10⁸. These results show that when a temperature of 70°C is reached and maintained for at least 2 min throughout a food there is a substantial reduction in the numbers of L. monocytogenes . The survival of this organism during microwave heating when temperatures of over 70°C are reported is probably due to uneven heating by microwave ovens resulting in the presence of cold spots in the product. The heat resistance of L. monocytogenes is comparable with that of many other non-sporing mesophilic bacteria.     

Factors affecting the growth of Listeria monocytogenes on minimally processed fresh endive

The influence of various factors on the fate of Listeria monocytogenes on cut leaves of broad-leaved endive has been studied. Factors considered were temperature, characteristics of the leaves (age, quantity and quality of the epiphytic microflora) and characteristics of the L. monocytogenes inoculum (concentration, strain). The increases in numbers of L. monocytogenes were lower than those of the aerobic mesophilic microflora at 3°, 6°, 10° and 20°C. Doubling times of the populations of L. monocytogenes were in the same order of magnitude as those of aerobic bacteria at 10° and 20°C, but longer at 3° and 6°C. There were positive significant correlations between growth of L. monocytogenes and populations of aerobic bacteria, and between growth of L. monocytogenes and extent of spoilage on the leaves.
Of 225 bacteria isolated from the leaves, 84% were identified as fluorescent pseudomonads; there was no difference in the species isolated from leaves that showed a low growth of L. monocytogenes and leaves that showed a high growth of L. monocytogenes. Populations of L. monocytogenes increased faster during the first 2 and 4 d of storage at 10°C on leaves inoculated with 10–10³ cfu g^-1 than on leaves inoculated with about 10⁵ cfu g^-1, but the population reached after 7 d was lower. The behaviour of L. monocytogenes was similar among the three strains tested.     

The lethal effect of carrot on Listeria species

When shredded or sliced carrots were inoculated with Listeria monocytogenes the number of viable listerias decreased rapidly. On carrot slices stored at 8°C there was a decrease after 3 d followed by an increase, after 7 d, to numbers similar to those present initially. The numbers of spoilage micro-organisms increased throughout storage at 8°C. Carrots macerated in a Stomacher Lab Blender also showed an antilisterial activity which resulted in a decrease in number of viable bacteria and in sublethal damage. The effect was shown by five carrot cultivars and acted on nine strains of L. monocytogenes and single strains of L. innocua, L. ivanovii, L. seeligeri, L. welshimeri . This antilisterial effect was heat-labile, was inactivated after a few hours at 4°C or at 30°C and was active over the pH range 5.8 to 7.0. Maceration of carrots in an Atomix blender for 1 min or in liquid nitrogen destroyed the antilisterial activity.     

Inhibition of Listeria monocytogenes and Listeria innocua by hexanoic and octanoic acids

Hexanoic acid and octanoic acid inhibited growth of 10 strains of Listeria monocytogenes and two strains of L. innocua at pH 5·0 and pH 5·5 and 20°C. Octanoic acid was more inhibitory than hexanoic acid and both were more inhibitory at pH 5·0 than at pH 5·5. The minimum inhibitory concentrations (MICs) were comparable with the concentrations of these acids that have been reported in Danish Blue cheese, where they were probably formed by the metabolism of Penicillium roquefortii . Thus hexanoic and octanoic acids may contribute to the inhibition of listerias in some cheeses.     

Survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer

The survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer was investigated. Raw milk was inoculated with different initial concentrations of L. monocytogenes and heated at temperatures ranging from 69 degrees to 73 degrees C. Listerias were not isolated from any of the milk samples immediately after thermal treatment. They were isolated, however, from 46.6% of heated samples (none from samples heated at 73 degrees C) after variable periods at refrigeration temperature. The results suggest that a low number of listerias survive some thermal treatments, but a cold enrichment is necessary to repair the thermally injured cells and detect these organisms in milk. The importance of the isolation technique in the recovery of listerias from pasteurized milk samples is discussed.     

Physicochemical surface properties of five Listeria monocytogenes strains from a pork-processing environment in relation to serotypes, genotypes and growth temperature

Physicochemical surface properties, related to electrostatic, van der Waals and Lewis acid–base interactions, of five Listeria monocytogenes strains isolated from pork-processing environments were determined after two subcultures at 37 °C and a final culture at three temperatures: 37, 10 and 4 °C. Three strains (Lm1, Lm114 and Lm191) were genetically related while two were unrelated (Lm25 and Lm74) according to Apa I-macrorestriction and pulsed-field gel electrophoresis (PFGE) typing.
Listeria monocytogenes cell surfaces were generally negatively charged regardless of pH and tended to be hydrophilic due to a basic character. However, variable physicochemical surface properties of the five Listeria monocytogenes isolates were observed after growth at 37 °C. After growth at 10 °C, the three genetically related isolates exhibited similar surface properties and were slightly more hydrophilic and basic than the others. After growth at 4 °C, the five isolates displayed the same weak affinity for all kinds of solvents and low electrophoretic mobility values.
A sharp decrease of temperature and subsequent growth of various Listeria monocytogenes strains resulted in loss of the physicochemical surface property variability, which may suggest the role of common chill adaptation mechanisms affecting surface properties.     

Effect of growth temperature on virulence of strains of Listeria monocytogenes in the mouse: evidence for a dose dependence

Growth of Listeria monocytogenes at 4°C significantly increased its virulence for mice by the intravenous route and the effect was dose-dependent. Virulence was apparent only at a dose of about or above 10⁴ viable listerias. At slightly lower doses of about 10³, no such effect was observed. Growth at 4°C did not increase the virulence of the strains for mice by oral-gastric challenge when given at doses of approximately 10¹⁰.     

Thermal inactivation of Listeria monocytogenes during a process simulating temperatures achieved during microwave heating

Conventional heating was used to expose cells of Listeria monocytogenes, either in broth or in situ on chicken skin, to the mean times and temperatures that are achieved during a 28 min period of microwave cooking of a whole chicken. Heating L. monocytogenes by this method in culture broth resulted in a reduction in viable cell numbers by a factor of greater than 10(6) upon reaching 70 degrees C. Simulated microwave cooking of L. monocytogenes in situ, on chicken skin, resulted in more variability in the numbers of survivors. Heating for the full cook time of 28 min, however, resulted in a mean measured temperature of 85 degrees C and no surviving listerias were detected. This indicated a reduction in viable numbers of greater than 10(6). To reduce temperature variation, cells were heated on skin in a submerged system in which exposure to 70 degrees C for 2 min resulted in a reduction in viable cell numbers of all strains of listerias tested of between 10(6) and 10(8). These results show that when a temperature of 70 degrees C is reached and maintained for at least 2 min throughout a food there is a substantial reduction in the numbers of L. monocytogenes. The survival of this organism during microwave heating when temperatures of over 70 degrees C are reported is probably due to uneven heating by microwave ovens resulting in the presence of cold spots in the product. The heat resistance of L. monocytogenes is comparable with that of many other non-sporing mesophilic bacteria.     

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.     

Growth of Listeria monocytogenes at refrigeration temperatures

The growth of three strains of Listeria monocytogenes at refrigeration temperatures (-0.5 to 9.3°C) in chicken broth and/or UHT milk was determined using a rocking temperature gradient incubator. Minimum growth temperatures ranged from -0.1 to -0.4°C for the three strains. Lag times of 1–3 d and 3 to >34 d were observed with incubation at 5 and 0°C respectively. Corresponding generation times ranged from 13–24 h at 5°C and 62–131 h at 0°C. The type of culture medium had an influence on both the rate and extent of growth. Incubation of cultures at 4°C before inoculation caused a marked reduction in the lag time when compared with cultures which had been previously incubated at 30°C.     

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.     

Heat resistance and growth of Salmonella enteritidis, Listeria monocytogenes and Aeromonas hydrophila in whole liquid egg

Salmonella enteritidis ATCC,13067, Listeria monocytogenes ATCC,19116 and Aeromonas hydrophila ATCC,7965 strains were evaluated for growth and thermal resistance in liquid whole egg (LWE). Each strain grew well in LWE at temperatures between 4 and 30 degrees C, except S. enteritidis which grew weakly at 4 and 10 degrees C. Maximum populations for each strain increased with increasing growth temperature. The thermal destruction of each strain was determined in six liquid products. The egg products used were LWE, LWE with 5, 10 and 15% NaCl and LWE with 5 and 10% sucrose. L. monocytogenes tended to be more heat resistant than S. enteritidis and A. hydrophila. The highest kill rates were noted in LWE, while survival was best in those products supplemented with NaCl. Radiation D10 values of strains in LWE were 0.18, 0.39 and 0.49 kGy for A. hydrophila, S. enteritidis and L. monocytogenes, respectively.     

Effect of temperature on sulphate reduction, growth rate and growth yield in five psychrophilic sulphate-reducing bacteria from Arctic sediments

Five psychrophilic sulphate-reducing bacteria (strains ASv26, LSv21, PSv29, LSv54 and LSv514) isolated from Arctic sediments were examined for their adaptation to permanently low temperatures. All strains grew at −1.8°C, the freezing point of sea water, but their optimum temperature for growth ( T _opt) were 7°C (PSv29), 10°C (ASv26, LSv54) and 18°C (LSv21, LSv514). Although T _opt was considerably above the in situ temperatures of their habitats (−1.7°C and 2.6°C), relative growth rates were still high at 0°C, accounting for 25–41% of those at T _opt. Short-term incubations of exponentially growing cultures showed that the highest sulphate reduction rates occurred 2–9°C above T _opt. In contrast to growth and sulphate reduction rates, growth yields of strains ASv26, LSv54 and PSv29 were almost constant between −1.8°C and T _opt. For strains LSv21 and LSv514, however, growth yields were highest at the lowest temperatures, around 0°C. The results indicate that psychrophilic sulphate-reducing bacteria are specially adapted to permanently low temperatures by high relative growth rates and high growth yields at in situ conditions.     

Influence of temperature on biofilm formation by Listeria monocytogenes on various food-contact surfaces: relationship with motility and cell surface hydrophobicity

Aims:  To assess the ability of Listeria monocytogenes to form biofilm on different food-contact surfaces with regard to different temperatures, cellular hydrophobicity and motility.
Methods and Results:  Forty-four L. monocytogenes strains from food and food environment were tested for biofilm formation by crystal violet staining. Biofilm levels were significantly higher on glass at 4, 12 and 22°C, as compared with polystyrene and stainless steel. At 37°C, L. monocytogenes produced biofilm at significantly higher levels on glass and stainless steel, as compared with polystyrene. Hydrophobicity was significantly ( P  < 0·05) higher at 37°C than at 4, 12 and 22°C. Thirty (68·2%) of 44 strains tested showed swimming at 22°C and 4 (9·1%) of those were also motile at 12°C. No correlation was observed between swimming and biofilm production.
Conclusions:  L. monocytogenes can adhere to and form biofilms on food-processing surfaces. Biofilm formation is significantly influenced by temperature, probably modifying cell surface hydrophobicity.
Significance and Impacts of the Study:  Biofilm formation creates major problems in the food industry because it may represent an important source of food contamination. Our results are therefore important in finding ways to prevent contamination because they contribute to a better understanding on how L. monocytogenes can establish biofilms in food industry and therefore survive in the processing environment.     

Growth rate and physiology of Yersinia enterocolitica; influence of temperature and presence of the virulence plasmid

The effect of temperature on the growth rate, protein pattern and fatty acid composition of Yersinia enterocolitica strain W22703 pYV⁺, its plasmidless isogenic derivative W22703 pYV^- and four recent field isolates was examined.
The growth rate was clearly influenced by presence or absence of the virulence plasmid: pYV^- strains grew consistently faster than pYV⁺ strains. This difference in growth rate was high at 30–35°C, moderate at 1–10°C and 25°C, but hardly significant at 15–20°C.
Increasing the growth temperature above 25°C resulted in the induction of the 220 kDa virulence plasmid-encoded Yop1 protein. In the 1–20°C range no obvious temperature- or plasmid-related differences in protein patterns could be detected.
The fatty acid composition showed a clear temperature-dependent change: with all strains the degree of saturation was low at 1°C and gradually increased with raising temperatures. All strains had similar fatty acid patterns, except one of the field isolates which showed aberrant C16 : 1 and cyclic fatty acid contents in the 5–25°C and 15°C ranges respectively. With strain W22703, the presence or absence of the virulence plasmid did not significantly alter the fatty acid pattern.     

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.     

Minimum water activity requirements for the growth of Listeria monocytogenes

The ability of five strains of Listeria monocytogenes to initiate growth at five different temperatures in brain heart infusion (BHI) broth adjusted to various water activity ( a _w) values with either sodium chloride (NaCl), sucrose or glycerol was investigated. Glycerol was the least toxic of the three solutes studied, with three of five strains of L. monocytogenes capable of growing in BHI broth adjusted with glycerol to an a w value of 0.90 at 30 C, compared to a w minima of 0.93 and 0.92 in broth adjusted with sucrose and sodium chloride, respectively. The minimum a w value required for growth generally increased as the incubation temperature decreased. Listeria monocytogenes appeared to tolerate glycerol and NaCl best when growing at 30 and 15°C, respectively, while for sucrose, temperature did not appear to influence growth of the organism. Listeria monocytogenes is one of the few food-borne pathogens that can grow at an a w value below 0.93.     

Heat resistance of Listeria: strain differences and effects of meat type and curing salts

The heat resistances of 27 strains of Listeria monocytogenes and two strains of L. innocua were compared in broth heated at 57°C. No strain was exceptionally resistant. The heat resistance of a representative isolate of L. monocytogenes was compared in fresh and cured beef and chicken, and an equation was derived to predict the time necessary to achieve a '7D' inactivation at temperatures between 50 and 70°C.