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.     

Survival of Listeria monocytogenes in yogurt during storage at 4°C

Cows' milk was inoculated with ca 10³and 10⁷cfu/ml Listeria monocytogenes. After fermentation at 42°C for 0–5 h, the yogurt was stored at 4°C. Low and high inocula survived for 48 h and 7 d, respectively; L. monocytogenes cells were not detectable by direct plating or cold-enrichment after 5 and 15 d, respectively. In low inoculum samples, initial pH at the time of refrigeration was 4·9; the final pH at the time of last sampling was 4·2. In the samples with high inoculum the pH decreased from 5·0 to 4·2.     

Effect of pre-incubation pH on the growth characteristics of Aeromonas hydrophila at 5°C, as assessed by two methods

Two strains of Aeromonas hydrophila (the type strain ATCC 7966 and a food-derived strain JAH4) were pre-incubated at 5°C in Brain Heart Infusion (BHI) broth with pH adjusted to 6.0 or 7.0, and then incubated at the same temperature in BHI broth with pH adjusted to 6.0, 6.5, 7.0 and 7.5. Growth kinetics during incubation were determined by two methods: viable count (VC) and measurement of optical density (O.D.). Pre-incubation at different pH values did not significantly affect the maximum specific growth rates of the strains during incubation, but the lag phases were shorter after pre-incubation at pH 6.0 than at pH 7.0. The VC method was more sensitive than O.D. measurements for assessing lag phase.     

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.     

Growth of Aeromonas spp. at 4°C and related toxin production

Over the last few years Aeromonas spp. have been considered to be potential intestinal pathogens (Altwegg & Geiss 1989). Recently, detailed evidence has linked a strain from contaminated shrimp with a clinical isolate from a patient with diarrhoea and found them to be the same (Altwegg et al. 1991). It is now known that Aeromonas spp. are present in many foods, especially those of animal origin (Palumbo et al. 1989). Furthermore, previous studies have shown that these organisms are quite capable of growing at refrigeration temperatures. For example, Palumbo et al. (1985) found that seven out of 14 strains showed significant growth within 14 d at 5°C. However, most studies have only examined a relatively small number of strains, which were not speciated and were simply called A. hydrophila.
In this paper we examine the growth of 36 strains of aeromonas (divided into A. hydrophila, A. sobria and A. caviae ) at 4°C, and their ability to produce cyotoxins at 4°C, 30°C and 37°C.     

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.     

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.     

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.     

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.     

Effect of fatty acid supplementation on the lipid composition of Mycobacterium smegmatis ATCC 607, grown at 27° and 37°C

Mycobacterium smegmatis ATCC 607 was grown at 27 and 37°C, with and without exogenous unsaturated fatty acids, viz. elaidic, oleic and palmitoleic acids, added to the growth medium. The total lipid content of M. smegmatis ATCC 607 was lower at 27°C, and with added oleic acid, when compared with the controls, but higher in presence of palmitoleic acid. At 37°C no significant differences were noted in the total lipid content. In general, the total lipid content was lower with all of the fatty acid supplementations at both 27 and 37°C. The phosphatidylethanolamine content was slightly higher at 27°C in the presence of elaidic or palmitoleic acid, but was markedly lower with oleic acid supplementation at 37°C. The cardiolipin content was lower in the presence of any of the fatty acids at 27°C, and higher in the medium supplemented with elaidic or oleic acid at 37°C. The unsaturated to saturated fatty acids ratio was higher with palmitoleic acid supplementation at 27°C, but remained unchanged in cells grown at 37°C. The modifications in mycobacterial lipids are a reflection of the organism's ability to adapt to changing growth conditions.     

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.     

A study of the growth kinetics of Yersinia enterocolitica serotype O:3 in pure and meat culture systems

The growth kinetics of a virulence plasmid-bearing (P+) and a plasmid-cured (P−) strain of Yersinia enterocolitica serotype O:3 in pure and meat culture were investigated. Growth studies were carried out at 25 and 37 °C in supplemented phosphate-buffered saline, buffered peptone water , cefsulodin-irgasan-novobiocin broth base or supplemented broth base (CIN). The lag phase durations and growth rates under these conditions were determined by linear regression analysis. In pure culture, under most sets of equivalent conditions, P+ and P− strains had similar lag phase durations. However, under one set of conditions, i.e. CIN broth at 37 °C, the lag phase duration of the P+ strain was significantly longer than P−. In all but the most selective medium, P+ strains had slower growth rates than P− strains at 37 °C, probably due to the increased metabolic burden entailed in the maintenance of the virulence plasmid. In the most selective medium, i.e. CIN broth, P+ strains grew significantly faster than P−. This finding suggests that possession of virulence plasmid confers an enhanced ability to grow in the presence of selective agents. In meat cultures, both strains had longer lag phases than in equivalent pure cultures, with longer lag phases noted at 37 than at 25 °C. No significant differences were observed between the length of lag phases of P+ and P− strains in meat culture. Both strains of Y. enterocolitica displayed faster growth rates in meat cultures than in pure cultures, indicating that one or more components of meat enhanced the growth of this organism. The effects and interaction of incubation temperature, enrichment broth and meat on the growth kinetics of plasmid-bearing and plasmid-cured Y. enterocolitica strains are discussed.     

Effect of various gas atmospheres on the growth and survival of Campylobacter jejuni on beef

Pieces of fresh beef were inoculated with three strains of Campylobacter jejuni . The meat was then allocated to three treatments: (a) vacuum packaged, (b) packaged in an atmosphere of 20% CO₂+ 80% N₂, and (c) packaged into sterile Petri dishes in anaerobic cultivation boxes, which were filled with a gas mixture of 5% O₂+ 10% CO₂+ 85% N₂. The packaging material in the first two treatments was PA 80/PE 100–PE 100/PA 80/PE 100. The survival of Campylobacter cells was followed at 37°C, 20°C and 4°C for 48 h, 4 days and 25 days, respectively. At 37°C the counts of two Campylobacter strains increased in each package treatment for 48 h. At 20°C and at 4°C the counts of the same two strains decreased by 1 to 2 log units and 0.5 to 1 log unit, respectively, during storage. The survival of the two strains was about the same in all package treatments. The third strain was the most sensitive of the strains studied. At 37°C its numbers increased only in the optimal gas atmosphere; at 20°C the strain was not detectable after 24 to 48 h storage and at 4°C after 4 days storage. The aerobic plate counts were determined for all samples at the same time as Campylobacter counts. The high indigenous bacterial numbers of the meat samples did not appear to have a great effect on the survival or growth of campylobacters.     

The role of L-carnitine and glycine betaine in the survival and sub-lethal injury of non-growing Listeria monocytogenes cells during chilled storage

AIMS: To determine the role played by previous growth in the presence of osmolytes on the subsequent survival and sub-lethal injury of L. monocytogenes during long-term chilled storage in a model buffer system. METHODS AND RESULTS: Four Listeria monocytogenes strains were grown separately to stationary phase in Listeria minimal medium (DM) alone or in DM with 4% NaCl alone, or both these media supplemented with 1 mM L-carnitine and/or 1 mM glycine betaine. Cells were resuspended in phosphate buffered saline (pH 5.5) and stored for four weeks at 4 degrees C. Initially, and at weekly intervals, samples were plated on both Tryptic Soy Agar and Tryptic Soy Agar with 4% NaCl to determine total numbers and degree of sub-lethal injury in the populations. The numbers of cells within all strains after growth to stationary phase, except one which increased ( approximately 2 log cfu ml-1, P < 0.05) in the presence of NaCl, were not influenced significantly by previous growth conditions (P > 0.05). During subsequent chilled storage, however, numbers of all strains grown in the presence of NaCl remained constant while those grown in its absence decreased. The rate and magnitude of the decrease in cell numbers was strain dependent. The initial percentage of sub-lethal injury increased significantly in all strains when grown previously in the presence of L-carnitine (P < 0.05). During subsequent chilled storage sub-lethal injury increased for all strains in a manner that was strain dependent, but not related to the previous growth conditions. CONCLUSION: Previous growth in the presence of osmolytes of NaCl, but not osmolytes alone, increases the subsequent survival, but not percentage sub-lethal injury, of L. monocytogenes during subsequent chilled storage in buffer. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that risks associated with L. monocytogenes in chilled food may be influenced by the individual life histories of the cells.     

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.     

The effect of nisin on Listeria monocytogenes in culture medium and long-life cottage cheese

M.A.S.S. FERREIRA AND B.M. LUND. 1996. The sensitivity to nisin of 27 strains of Listeria monocytogenes , four of L. innocua and one of L. ivanovii was estimated at pH 6.8 and pH 5.5. Strains of L. monocytogenes showed differences in sensitivity which were not correlated with serotype. Strains of L. innocua were as resistant as the most resistant strains of L. monocytogenes , whereas the strain of L. ivanovii was relatively sensitive. Two of the most resistant strains of L. monocytogenes multiplied in aerated liquid medium adjusted to pH 5.0 with HCl, incubated at 20°C; nisin, 500 IU ml^-1, prevented multiplication and caused death. Following inoculation of a resistant strain into long-life cottage cheese, pH 4.6–4.7, the number of viable L. monocytogenes decreased approximately 10-fold during storage at 20°C for 7 d; addition of nisin, 2000 IU g^-1, to the cottage cheese increased the rate of inactivation to approximately a 1000-fold decrease in 3 d.     

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.     

Effect of Hyperbaric Carbon Dioxide Pressure on the Microbial Flora of Pork Stored at 4 or 14°C

Changes in the microbial flora of pork stored at 4 or 14°C were studied in 5 atm CO₂, 1 atm CO₂ or 1 atm air. The time needed for the total aerobic count at 4°C to reach 5 × 10⁶ organisms/cm² was about three times longer in 5 atm CO₂ than in 1 atm CO₂, and about 15 times longer in 5 atm CO₂ than in air. At 14°C there was no difference in growth rate between 5 atm CO₂ and 1 atm CO₂. No off-odour was detected after storage in 5 atm CO₂ for 14 d, but the pork in 1 atm CO₂ (6 d) was organoleptically unacceptable.
The predominant organisms on the pork from the processing line were: Flavobacterium spp., Acinetobacter calcoaceticus, Pseudomonas spp., Micrococcus spp. and Moraxella spp. After aerobic storage at 4°C (8 d) or 14°C (3 d) more than 90% of the flora consisted of Pseudomonas spp. At 4°C all Pseudomonas spp. were of the non-fluorescent type, whilst at 14°C 32% were Ps. putida and Ps. fluorescens. After storage in 1 atm CO₂ Lactobacillus spp. represented 66% of the flora at 14°C (6 d) and 100% at 4°C (40 d), with L. xylosus dominating. After storage in 5 atm CO₂ Lactobacillus spp. constituted the total flora at both temperatures with L. lactis (14°C) and L. xylosus (4°C) dominating.
It was concluded that high partial pressures of CO₂ have a considerable shelf-life prolonging effect by (i) selecting the microflora towards Lactobacillus spp. and (ii) reducing the growth rate of these Lactobacillus spp. The controlling and growth inhibitory effect of CO₂ was promoted by reduced temperatures.     

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.