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.     

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.     

Effect of salt concentration and temperature on survival of Legionella pneumophila

The effects of various concentrations of sodium chloride solutions (0·1%–3%) and different temperatures (4, 10, 20, 30 and 37 °C) on survival of Legionella pneumophila were investigated. It was found that at temperatures between 4 °C and 20 °C, Legionella organisms survived in salt solutions up to 3% NaCl. Only the combination of high temperatures, i. e. 30 °C and 37 °C, with NaCl concentrations over 1·5%, reduced cell numbers significantly. It was interesting to note that the addition of small amounts of NaCl (0·1%–0·5%) enhanced survival of Leg. pneumophila , suggesting a protective effect of NaCl. In order to obtain information about conditions encountered in the environment, the survival experiments were repeated in sterile sea water from the Baltic Sea and the North Sea. The marked bacterial die-off, especially at higher temperatures, was not observed in natural sea water. All these results indicate that Leg. pneumophila can survive in the marine environment.     

Characterization of probiotic carnobacteria isolated from rainbow trout (Oncorhynchus mykiss) intestine

Aims:  To characterize two probiotic carnobacterial isolates, Carnobacterium maltaromaticum (B26) and C. divergens (B33), derived from rainbow trout ( Oncorhynchus mykiss ) intestine.
Methods and Results:  Both cultures, which were able to colonize the fish gut mucosal layer, comprised nonsporogenous, nonmotile, Gram-positive, catalase and oxidase-negative rods. The growth of both carnobacteria occurred between 0 and 37°C, in 0–10% (w/v) NaCl and at pH 5–10. Specifically, strain B26 grew in nutrient broth supplemented with 15% (w/v) NaCl. The most abundant cellular fatty acid of both cultures was 9-octadecenoic acid (18 : 1 n -9) (B26 = 52·6%; B33 = 40·6%), which was characteristic of Carnobacterium . Both cultures were inhibitory to Aeromonas salmonicida , Aer. hydrophila , Streptococcus iniae and Vibrio anguillarum , and strain B33 inhibited Listeria monocytogenes . Both carnobacteria, which did not contain plasmids, produced inhibitory compounds against Gram-positive and Gram-negative bacteria.
Conclusions:  Both probiotic cultures, B26 and B33, had unique phenotypic characteristics and showed a broad spectrum of antibiotic resistance against varying pathogenic bacteria.
Significance and Impact of the Study:  The results of this study contribute to new information and significance of carnobacterial species.     

Survival of Listeria monocytogenes in sea water and effect of exposure on thermal resistance

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes , Scott A and an environmental strain KM, on exposure to sea water at 12·8 or 20·8 °C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T₉₀ values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0·6% yeast extract) at 12·8 and 20·8 °C were 61·7 and 69·2 h for L. monocytogenes Scott A, and 103·0 and 67·0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T₉₀ values at 12·8 and 20·8 °C were 60·6 and 56·9 h for L. monocytogenes Scott A, and 83·0 and 65·9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12·8 and 20·8 °C was 1·7 and 17·7%, respectively, while for KM the values were 19·0 and 1·6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 °C showed an apparent increase in heat resistance after exposure to sea water at 20·8 °C for 7 d ( D ₅₈= 2·64 min) compared with before exposure ( D ₅₈= 1·24). This increase in thermal resistance was not apparent at temperatures greater than 63 °C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different ( P > 0·05) over the temperature range tested (58–62 °C).     

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.     

Sodium chloride affects Listeria monocytogenes adhesion to polystyrene and stainless steel by regulating flagella expression

Aim:  To study the adhesion capability of seven strains of Listeria monocytogenes to polystyrene and stainless steel surfaces after cultivation at various NaCl concentrations.
Methods and Results:  Determination of growth limits indicated that all seven strains were able to grow in up to 11% NaCl in rain heart infusion and 3 g l⁻¹ yeast extract–glucose at 20°C, but no growth was detected at 15% NaCl. Adhesion of L. monocytogenes was estimated after 4-h incubation at 20°C in 96-well microtitre plates. Statistical results revealed no significant difference between adhesion to polystyrene and stainless steel although surface properties were different. Adhesion between 0% and 6% NaCl was not different, whereas adhesion at 11% NaCl was significantly lower. This discrepancy in adhesion was correlated with the down-regulation of flagella at 11% NaCl.
Conclusions:  Only high salinity levels, close to nongrowth conditions, repressed the expression of flagella, and consequently, decreased the adhesion capability of L. monocytogenes .
Significance and Impact of the Study:  Adhesion of L. monocytogenes to inert surfaces depends on environmental conditions that affect flagellum expression. High salinity concentrations would delay biofilm formation.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

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.     

The effect of slurry storage and anaerobic digestion on survival of pathogenic bacteria

The decline in viable numbers of Salmonella typhimurium, Yersinia enterocolitica and Listeria monocytogenes in beef cattle slurry is temperature-dependent; they decline more rapidly at 17°C than at 4°C. Mesophilic anaerobic digestion caused an initial rapid decline in the viable numbers of Escherichia coli, Salm. typhimurium, Y. enterocolitica and L. monocytogenes. This was followed by a period in which the viable numbers were not reduced by 90%. The T₉₀ values of E. coli, Salm. typhimurium and Y. enterocolitica ranged from 0.7 to 0.9 d during batch digestion and 1.1 to 2.5 d during semi-continuous digestion. Listeria monocytogenes had a significantly higher mean T₉₀ value during semi-continuous digestion (35.7 d) than batch digestion (12.3 d). Anaerobic digestion had little effect in reducing the viable numbers of Campylobacter jejuni.     

Thermal inactivation of Listeria monocytogenes during rapid and slow heating in sous vide cooked beef

T.B. HANSEN AND S. KNØCHEL. 1996. Heating at slowly rising temperatures is suspected to enhance thermotolerance in Listeria monocytogenes and, since anaerobic environments have been shown to facilitate resuscitation of heat-injured cells of this micro-organism, concern may arise about the possibility of L. monocytogenes surviving in minimally preserved products. The effect of rapid (> 10°C min^-1) and slow (0.3 and 0.6°C min^-1) heating on survival of L. monocytogenes in sous vide cooked beef was therefore examined at mild processing temperatures of 56, 60 and 64°C. No statistically significant difference ( P = 0.70) was observed between the tested heating regimes. Since the average pH of beef was low (5.6), and little or no effect was observed, a pH-dependency of heat shock-induced thermotolerance in L. monocytogenes is suggested to account for this result.     

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.     

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.     

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.     

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.     

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.C. The mean minimum temperature for L. monocytogenes was +1.1 0.3.C. The growth of non-haemolytic listerias was unobservable at +1.7 0.5.C. The L. monocytogenes strains grew at about 0.6°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 (+1.0.3°C) than the other common serovar 4b (+1.3 0.4°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.     

Developmental strategies of Koenigia islandica, a high-arctic annual plant

Seed germination, growth and flowering of the arctic-alpine annual Koenigia islandica were studied in controlled environment. Intact (unabraded) seeds germinated poorely at temperatures up to 18°C, with an optimum at 24°C (89% in 10 d). Scarified seeds germinated rapidly, and reached 100% germination in 3 d at 21°C, but no >40% germination occurred at 9 and 12°C, The seeds had no light requirement for germination, nor did fluctuating temperatures improve germination
Dry matter production was optimal at 12°C in both short day (SD) and long day (LD) conditions, but was markedly higher in LD than in SD at identical fluences at all temperatures except 21°C where the plants showed symptoms of severe heat stress. The temperature compensation point for net productivity was estimated to 24°C, and negative carbon balance at higher temperatures might be an important physiological mechanism limiting the distribution of K. islandica in Scandinavia.
Flowering was extremely rapid and independent of daylength, even in a high-arctic population from 79°N, In full summer daylight anthesis was reached 24 d after germination and seeds ripened after 36 d at 15°C, Days to anthesis varied little across the temperature range from 6 to 21°C, giving a linear decrease in the heat-sum requirement for the attainment of flowering with decreasing temperature.
It is concluded that conservative seed germination strategy, tininess and rapid development, low temperature optima for growth and reproduction, and daylength indifference of flowering are important adaptations for success of an annual plant in high-arctic and high-alpine environments, Daylength neutrality has facilitated the wide-latitudinal distribution of K. islandica. including the penetration of the species to the southern hemisphere.     

Thermal dependence of embryonic growth and development in brown trout

Fertilized eggs from a brown trout Salmo trutta population in northern Spain were incubated in the laboratory at 4, 6, 8, 10, 12, 14, 16 and 18° C. Developmental stage and embryo size were monitored by taking samples at regular intervals. Survival was maximal at 8 and 10° C and decreased at higher and lower temperatures. Despite starting development, no embryo hatched at 16 and 18° C, which suggests an upper thermal limit for development between 14 and 16° C. Time required to reach a given ontogenetic stage decreased with increasing temperature. Embryos incubated at lower temperatures were larger at 50% hatching, and these differences persisted throughout the subsequent embryonic period until the start of exogenous feeding. A comparison with previously published data indicates low interpopulation variability in thermal sensitivity of embryonic development, even in consideration of the great latitudinal range of the studies.     

Comparison of the growth of Listeria monocytogenes in unirradiated and irradiated cook-chill roast beef and gravy at refrigeration temperatures

Specific growth rates of two strains of Listeria monocytogenes in unirradiated and irradiated (2 kGy) roast beef and gravy stored at 5° and 10°C were found to be similar. However, exponential growth of L. monocytogenes after irradiation was preceded by an extended lag period of 6–9 d at 5°C and 3–4 d at 10°C, compared with lag periods of 1–2 d and <0.1 d in unirradiated beef and gravy stored similarly.     

Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C

The effect of mint ( Mentha piperita ) essential oil (0·5, 1·0, 1·5 and 2·0%, v/w) on Salmonella enteritidis and Listeria monocytogenes in a culture medium and three model foods; tzatziki (pH 4·5), taramosalata (pH 5·0) and pâté (pH 6·8), inoculated at 10⁷ cfu g^-1, at 4° and 10°C for ca 1 week was studied. In the culture medium supplemented with the essential oil, no growth was observed over 2 d at 30°C determined by a conductance method with a Malthus 2000 growth analyser. Salmonella enteritidis died in tzatziki in all treatments and declined in the other foods except for pâté at 10°C as judged with viable counts. Listeria monocytogenes populations showed a declining trend towards the end of the storage period but was increased in pâté. Mint essential oil antibacterial action depended mainly on its concentration, food pH, composition, storage temperature and the nature of the micro-organism.