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.     

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.     

The survival of Salmonella enterica serovar Typhimurium DT104 and total viable counts on beef surfaces at different relative humidities and temperatures

Aims:  The aim of this study was to investigate changes in Salmonella and total viable count (TVC) survival on beef carcass surfaces stored for 72 h under different combinations of relative humidity (i.e. RH 75% or 96%) and temperature (5°C or 10°C).
Methods and Results:  The influence of low water activity ( a _w) and temperature on the survival and growth of Salmonella enterica serovar Typhimurium DT104 and the aerobic mesophilic flora on meat pieces from different sites on beef carcasses was investigated, under controlled conditions (75% or 96% RH; 5 or 10°C) in an environmental cabinet. Salmonella counts declined during storage at low a _w (75% RH) conditions at 5°C or 10°C. Salmonella counts increased during storage at high a _w (96% RH) at 10°C only. At 5°C, TVCs increased during storage at high a _w, but not at low a _w. TVCs increased on all samples from carcasses stored at high or low a _w at 10°C, except those samples taken from areas of surface fat.
Conclusions:  This suggests that substrate composition dictates growth rates under low a _w conditions. The results are discussed in terms of the possible protective effects of substrate osmolyte accumulation in bacterial survival and/or growth.
Significance and Impact of the Study:  The data obtained in this study provides useful insights on the influence of a _w and temperature on pathogen survival on meat surfaces at chill temperature.     

Effect of curli expression and hydrophobicity of Escherichia coli O157:H7 on attachment to fresh produce surfaces

Aim: To investigate the effect of curli expression on cell hydrophobicity, biofilm formation and attachment to cut and intact fresh produce surfaces. Methods and Results: Five Escherichia coli O157:H7 strains were evaluated for curli expression, hydrophobicity, biofilm formation and attachment to intact and cut fresh produce (cabbage, iceberg lettuce and Romaine lettuce) leaves. Biofilm formation was stronger when E. coli O157:H7 were grown in diluted tryptic soy broth (1 : 10). In general, strong curli‐expressing E. coli O157:H7 strains 4406 and 4407 were more hydrophobic and attached to cabbage and iceberg lettuce surfaces at significantly higher numbers than other weak curli‐expressing strains. Overall, E. coli O157:H7 populations attached to cabbage and lettuce (iceberg and Romaine) surfaces were similar (P > 0·05), indicating produce surfaces did not affect (P < 0·05) bacterial attachment. All E. coli O157:H7 strains attached rapidly on intact and cut produce surfaces. Escherichia coli O157:H7 attached preferentially to cut surfaces of all produce types; however, the difference between E. coli O157:H7 populations attached to intact and cut surfaces was not significant (P > 0·05) in most cases. Escherichia coli O157:H7 attachment and attachment strength (S_R) to intact and cut produce surfaces increased with time. Conclusions: Curli‐producing E. coli O157:H7 strains attach at higher numbers to produce surfaces. Increased attachment of E. coli O157:H7 on cut surfaces emphasizes the need for an effective produce wash to kill E. coli O157:H7 on produce. Significance and Impact of the Study: Understanding the attachment mechanisms of E. coli O157:H7 to produce surfaces will aid in developing new intervention strategies to prevent produce outbreaks.     

Microbial population profiles of the microflora associated with pre- and postharvest tomatoes contaminated with Salmonella typhimurium or Salmonella montevideo

Aims:  To determine the microflora profiles of pre- and postharvest tomatoes contaminated with Salmonella montevideo or S. typhimurium DT104.
Methods and Results:  Salmonella montevideo or S. typhimurium was inoculated onto the flowers of tomato plants with the microflora of the subsequent fruit examined using a combination of Source Carbon Utilization and 16S rDNA-PCR profiling. From 16S rDNA profiles it was evident that tomatoes derived from Salmonella inoculated plants harboured a different microbial population compared to nontreated controls. The same result was observed for tomatoes inoculated at postharvest and subsequently stored for 14 days at 15°C. From sequencing analysis it was found that tomatoes derived from Salmonella inoculated plants but testing negative for the enteric pathogen, frequently harboured Enterobacter and Bacillus spp. In contrast, both bacterial types were not found associated with tomatoes testing positive for Salmonella.
Conclusions:  Salmonella introduced onto tomatoes at pre- or postharvest alters the composition of the microbial community. The presence of Enterobacter and Bacillus spp negatively affects the persistence of Salmonella on preharvest tomatoes.
Significance and Impact of the Study:  Salmonella appears to modify rather than become integrated into the microbial communities associated with tomatoes. Yet, the presence of antagonistic bacteria appears to reduce the persistence of the enteric pathogen.     

Involvement of putative chemical wound signals in the induction of phenolic metabolism in wounded lettuce

Cutting leaves of Romaine lettuce ( Lactuca sativa L. cv. Longifolia) produces a wound signal that induces the synthesis of phenylalanine ammonia lyase (PAL, EC 4.3.1.5) and the accumulation of phenolic compounds in cells up to 2 cm from the site of injury, and tissue browning near the site of injury. The response of leaves within a head of Romaine lettuce to putative chemical wound signals [abscisic acid (ABA), jasmonate (JA) and methyl jasmonate (MeJA)] differed significantly with leaf age. Exposure of harvested heads of lettuce to ABA, JA, MeJA, or salicylic acid (SA) did not induce changes in PAL activity, the concentration of phenolic compounds or browning in mature leaf tissue that was similar to the level induced by wounding. Methyl jasmonate applied as vapour (10, 100 or 1000 µl kg⁻¹ FW), or as an aqueous spray or dip (0.01–100 µ M ) at 5 or 10°C did not produce an effect on PAL activity or browning that differed significantly from the untreated controls. In contrast, JA, MeJA and SA did induce elevated levels of PAL activity in younger leaves. However, the levels induced were far lower than those induced by wounding. Wound induced phenolic metabolism in mature leaves appears to be induced by different signals than those functioning in young leaves.     

Survival of Salmonella on refrigerated chicken carcasses and subsequent transfer to cutting board

Objective:  To determine the effect of refrigeration time and temperature on Salmonella cell numbers on inoculated chicken carcasses and their transfer to a plastic cutting board.
Methods and Results:  The survival of Salmonella on chicken skin and the transfer to a plastic cutting board when exposed to different refrigeration temperatures (2, 6 or 8°C) for 9 days were the two main issues on which this work focused. Two scenarios were carried out to ascertain these effects: carcasses treated with a decontaminating acetic acid solution and untreated carcasses. All of the contaminated carcasses remained contaminated after 9 days of refrigeration. However, on untreated samples, while Salmonella numbers increased almost 1·5 log at 8°C, the pathogen numbers decreased about 1 log at 2 and 6°C. On acid-treated samples, cell numbers slightly decreased at all of the temperatures studied. Temperature did not affect salmonellae transfer to the cutting board, but time did. Acid decontamination increased cell numbers transferred to the cutting board compared with untreated samples.
Conclusion:  Proper refrigeration at low temperatures did not allow Salmonella numbers to rise, regardless of which carcasses had been, or had not been, acid treated. Despite the fact that the rate of transfer was not affected by temperature, the acid treatment detached Salmonella cells from the chicken skin and, therefore, the probability of greater cross-contamination should be studied further.
Significance and Impact of the Study:  The results of this study may provide better information about the refrigeration conditions for fresh chicken storage and also determine if these, along with acetic acid decontamination of broiler chicken, would affect the pathogen transfer to a cutting board.     

Oviposition site selection by Mansonia mosquitoes on water lettuce

Abstract. 1. Viable Mansonia egg masses occur in nature on both the upper and under surfaces of Pistia stratiotes (L.) leaves. Upper-surface masses were from Mansonia dyari Belkin, Heinemann & Page, but lower-surface oviposition was attributable to both M. dyari and M. titillans Walker.
2. Upper-surface egg masses were often out of water but were concentrated near a wet-dry stain line on the leaf. Lower-surface masses were laid underwater within 3 mm of the leaf edge, except for those oviposited through fenestrations caused by insect damage.
3. Oviposition varied seasonally and masses were most numerous between August and December when water lettuce plants were large and crowded. Egg masses were clumped on plants and leaves and concentrated on mature or mature-old leaves that subtended mean angles of 28–34° from the horizontal.
4. On intact plants in cages, M. dyari laid egg masses on both leaf surfaces in approximately the same proportions as observed in nature. On detached leaves floating flat on the water, M. dyari laid all masses on under surfaces.
5. At 20 and 25°C, egg development of M. dyari required 17.4 and 8.8 days (means), these being significantly longer than the mean times for M. titillans. Some larvae from eggs that hatched in the absence of water survived 72 h in the eggshell.
6. Upper-surface oviposition by M. dyari may be an adaptation to the crowded growth of water lettuce, the mosquito's favoured host plant, whose leaves are largely out of water. Leaf ageing and subsidence cause submergence of most upper-surface eggs by the time of hatching.     

Extracellular protectants produced by Clostridium perfringens cells at elevated temperatures

Aim:  The mechanisms of adaptation of Clostridium perfringens to high temperatures are not well understood. In this work, the involvement of extracellular compounds in protection to heat was determined.
Methods and Results:  Cells were grown in fluid thioglycollate medium or chicken broth. When mid-log phase was reached, they were heat-shocked at 50°C for 30 min. Then cultures were centrifuged and supernatants were transferred to nonshocked cells. Heat tolerance of these cells was performed at 55°C. Viable cells were determined. In some cases, supernatants were heated at 65°C or 100°C or treated with trypsin. Supernatants were fractionated and PAGE was made of fractions showing heat-protective activity. When C. perfringens was exposed to a heat shock at 50°C, extracellular factors were found in the culture supernatant that provided protection to cells not exposed to a heat shock. The extracellular factors were sensitive to heat and trypsin treatment suggesting a protein component. SDS-PAGE analysis of supernatant fractions from heat-treated cells revealed two induced proteins (56 and 125 kDa) that could be involved in heat tolerance.
Conclusion:  In this work, the presence and thermoprotective activity of extracellular factors produced by C. perfringens under a heat shock was demonstrated.
Significance and Impact of the Study:  The detection of thermoprotective extracellular factors of C . perfringens will aid in our understanding of the physiology of survival of C. perfringens in foods.     

Heat resistance of Cronobacter species (Enterobacter sakazakii) in milk and special feeding formula

Aim:  To determine D - and z -values of Cronobacter species ( Enterobacter sakazakii ) in different reconstituted milk and special feeding formula and the effect of reconstitution of powdered milk and special feeding formula with hot water on the survival of the micro-organism.
Methods and Results:  Five Cronobacter species (four C. sakazakii isolates and C. muytjensii ) were heated in reconstituted milk or feeding formula pre-equilibrated at 52–58°C for various times or mixed with powdered milk or feeding formula prior to reconstitution with water at 60–100°C. The D -values of Cronobacter at 52–58°C were significantly higher in whole milk (22·10–0·68 min) than in low fat (15·87–0·62 min) or skim milk (15·30–0·51 min) and significantly higher in lactose-free formula (19·57–0·66 min) than in soy protein formula (17·22–0·63 min). The z -values of Cronobacter in reconstituted milk or feeding formula ranged from 4·01°C to 4·39°C. Water heated to ≥70°C and added to powdered milk and formula resulted in a > 4 log₁₀ reduction of Cronobacter .
Conclusions:  The heat resistance of Cronobacter should not allow the survival of the pathogen during normal pasteurization treatment. The use of hot water (≥70°C) during reconstitution appears to be an effective means to reduce the risk of Cronobacter in these products.
Significance and Impact of the Study:  This study supports existing data available to regulatory agencies and milk producers that recommended heat treatments are sufficient to substantially reduce risk from Cronobacter which may be present in these products.     

Wound-induced PAL activity is suppressed by heat-shock treatments that induce the synthesis of heat-shock proteins

Wounding lettuce leaves induces the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the accumulation of phenolic compounds, and subsequent tissue browning. A brief heat-shock at 45°C reduces the rise in wound-induced PAL, the accumulation of phenolic compounds, and tissue browning. The activity of PAL measured 24 h after wounding and the content of phenolic compounds (absorbance of methanol extract at 320 nm) measured 48 h after wounding was highly correlated (R² > 0.90) in tissue developing the normal wound response and in tissue subjected to 0–180 s of heat-shock after wounding. The synthesis of a unique set of proteins called heat-shock proteins (hsps) is induced by these heat-shock treatments. Western-blot analyses of proteins isolated from wounded and heat-shocked Iceberg and Romaine lettuce mid-rib leaf tissue was done using antibodies against hsp 23. Only those heat-shock treatments that were effective at inducing the synthesis of hsp 23 were effective in reducing the activity of PAL induced by wounding and the subsequent accumulation of phenolic compounds. Hsps induced in non-wounded, whole leaves by exposure to 45°C for 150 s did not significantly interact with PAL previously synthesized in non-heat-shocked wounded leaves to limit its activity. The preferential synthesis of hsps over that of wound-induced PAL, rather than the presence of hsps, may be responsible for the ability of a heat-shock treatment to reduce the wound-induced increase in PAL activity. Our results support this novel concept, and the possibility that heat-shock treatments can have significant physiological effects on the response of the tissue to other stresses, not because of the specific genes they induce or repress, or the products they cause to be synthesized, but by their secondary action of influencing the synthesis of other proteins (e.g. PAL) by the suppression of non-hsps protein synthesis.     

Use of episcopic differential interference contrast microscopy to identify bacterial biofilms on salad leaves and track colonization by Salmonella Thompson

Zoonotic pathogens such as Salmonella can cause gastrointestinal illness if they are ingested with food. Foods such as salads pose a greater risk because they are consumed raw and have been the source of major outbreaks of disease from fresh produce. The novel light microscopy methods used in this study allow detailed, high resolution imaging of the leaf surface environment (the phyllosphere) and allow pathogen tracking. Episcopic differential interference contrast microscopy coupled with epifluorescence was used to view the natural microflora in situ on salad leaves and their topographical distribution. Fluorescent nucleic acid staining was used to differentiate between bacterial colonists and inorganic debris. Salmonella enterica serovar Thompson expressing green fluorescent protein was inoculated onto individual spinach leaves for 24 h at 22°C in order to observe spatial and temporal patterning of colonization on the two surfaces of each leaf under different osmotic conditions. The results obtained show that salad leaves are host to high numbers of bacteria, typically 10⁵ per square millimetre. Cells are present in complex three-dimensional aggregations which often have a slimy appearance, suggesting the presence of biofilms. Washing of the leaves had little effect on the number of adherent pathogens, suggesting very strong attachment. Episcopic differential interference contrast microscopy is a rapid alternative to both scanning electron microscopy and confocal laser scanning microscopy for visualizing leaf topography and biofilm formation in the natural state.     

Contact with the host plant enhances aphid survival at low temperatures

1. The hypothesis, suggested by previous studies, that host plant contact reduces the cold tolerance of anholocyclic aphids was tested under laboratory conditions. Adult and first-instar Rhopalosiphum padi (L.) were exposed to temperatures of 0, –5 and –10 °C on intact plants, excised leaves and in the absence of contact with plant material.
2. Median lethal time (LT₅₀) values at all three temperatures indicate that aphids exposed in association with plant material survive longer than aphids that have no contact with their host plant. The difference in survival was most pronounced at –10 °C. Therefore, the above hypothesis is rejected for aphids on cereals because host plant contact apparently enhances cold tolerance.
3. Exposure on excised leaves also enhanced aphid survival at low temperature but was less effective than the intact plant. This suggests that plant quality as well as the presence or absence of plants is important in the cold tolerance of aphids on cereals.     

Survival of enteric bacteria and coliphage MS2 in pure human urine

Aims:  The survival of Escherichia coli , Salmonella enterica serovar Typhimurium, Enterococcus faecalis and coliphage MS2 was studied in stored, fresh and diluted (1 : 1) human urine at 15 and 30°C.
Methods and Results:  Survival rate was studied by the plate count method. All the organisms showed rapid inactivation in stored urine, but they survived better in diluted and fresh urine. The high pH level and temperature were the major factors found to influence the survival of the micro-organisms with the survival rate being higher at 15°C than at 30°C.
Conclusions:  The destruction of all micro-organisms in stored urine required <1 week at 30°C. Thus, the storage of urine is a useful way to reduce the risk of contamination while using urine as a fertilizer.
Significance and Impact of the Study:  The urine fertilization is aimed for the developing countries and the high temperatures in these countries may hasten the destruction of micro-organisms in urine. On the contrary, a higher survival rate of these organisms in fresh and diluted urine is a public health concern because the dilution of urine with water is likely to happen during flushing.     

The formation of spores in biofilms of Anoxybacillus flavithermus

Aims:  To examine the rate and the extent of spore formation in Anoxybacillus flavithermus biofilms and to test the effect of one key variable – temperature – on spore formation.
Methods and Results:  A continuous flow laboratory reactor was used to grow biofilms of the typical dairy thermophile A. flavithermus (strain CM) in skim milk. The reactor was inoculated with either a washed culture or a spore suspension of A. flavithermus CM, and was run over an 8·5 h period at three different temperatures of 48, 55 and 60°C. Change in impedance was used to determine the cell numbers in the milk and on the surface of the stainless steel reactor tubes. The biofilm developed at all three temperatures within 6–8 h. Spores formed at 55 and 60°C and amounted to approx. 10–50% of the biofilm. No spores formed at 48°C.
Conclusions:  The results suggest that both biofilm formation and spore formation of A. flavithermus can occur very rapidly and simultaneously. In addition, temperature variation has a considerable effect on the formation of spores.
Significance and Impact of the Study:  This information will provide direction for developing improved ways in which to manipulate conditions in milk powder manufacturing plants to control biofilms and spores of A. flavithermus .     

Changes in Escherichia coli O157:H7 numbers during holding on excised lean, fascia and fat beef surfaces at different temperatures

Aim:  To investigate changes in Escherichia coli O157:H7 numbers on excised beef carcass surfaces over 72 h at different temperatures.
Methods and Results:  Excised lean meat, fascia and fat were inoculated with E. coli O157:H7 and held in an environmental chamber for 72 h, at air speed 0·5 m s⁻¹, relative humidity (RH) 90%, and temperatures 4, 8 and 12°C. On lean, pathogen counts increased significantly at 12°C. On fascia, significant reductions in counts occurred at 4 and 8°C. Pathogen numbers were significantly reduced on fat at 4, 8 and 12°C (64 h). Counts on fat were significantly less at all temperatures, compared to lean or fascia and surface water activity, a_w, decreased significantly over time on fat at 4°C. Significant decreases in surface pH values were recorded on all meat substrates.
Conclusions:  The survival of E. coli O157:H7 varied in relation to the meat substrate and the holding temperature. Reductions in counts on fat surfaces appeared to be related to low surface a_w values. No relationship between pathogen survival and surface pH was established.
Significance and Impact of the Study:  The use of excised meat pieces in an environmental cabinet offers a more flexible approach to determining the use of different chilling regimes in the production of safe meat.     

Sanitation of faeces from source-separating dry toilets using urea

Aim:  To develop a reliable and simple method to produce safe fertilizers from human excreta using urea for sanitation of faeces.
Methods and Results:  Urea was added to faecal matter (17% dry matter) at concentrations of 0·5–2% (w/w) and inactivation of Salmonella enterica subspecies 1 serovar Typhimurium (Salm. Typhimurium), Enterococcus spp . and the Salm. Typhimurium bacteriophage 28B was monitored at 14, 24 and 34°C. Urea additions enhanced inactivation and inactivation rates were positively related to increasing NH₃ (aq) concentration and temperature. Salm. Typhimurium was the most sensitive of the organisms studied, while Enterococcus spp. showed more persistence, especially at lower temperatures. The bacteriophage was the most resistant organism studied.
Conclusions:  Salmonella reduction levels that meet requirements for safe reuse of faeces as fertilizer (i.e. 6 log₁₀ reduction) can be achieved for 1% urea within 2 months at 14°C or within 1 week at 24°C and 34°C.
Significance and Impact of the Study:  The relationships between organism inactivation rates and temperature, ammonia and pH were identified. Urea treatment proved to be a robust and efficient option for safe recycling of plant nutrients.     

Antimicrobial mortar surfaces for the improvement of hygienic conditions

Aims:  To evaluate the effectiveness of various antimicrobial mortar formulations in inhibiting the growth of a selection of pathogens of environmental and hygienic concern.
Methods and Results:  Mortar prisms containing triclosan-incorporated fibres or different concentrations of silver copper zeolites were incubated with Escherichia coli , Listeria monocytogenes , Salmonella enterica or Staphylococcus aureus at 4 or 20°C for 24 h. From plate counting, a substantial bactericidal effect (>4 log units) could only be observed for the mortar specimens containing more than 3% zeolites on cement weight base, the effect being more pronounced at 20°C compared to 4°C. No inhibitory effect could be observed for mortar specimens containing antimicrobial fibres. Adenosinetriphosphate (ATP) measurements allowed for a rapid indication of the occurrence of antimicrobial activity.
Conclusions:  In order to obtain a bactericidal effect on mortar surfaces, concentrations of silver copper zeolites of more then 3% are required.
Significance and Impact of the Study:  To our knowledge, this is the first study in which the effectiveness of various antimicrobial mortar mixtures towards the inhibition of pathogens has been evaluated in a quantitative way. Antimicrobial concrete mixtures can be used for the improvement of the hygienic conditions in a variety of environments.     

Intergenerational acclimation in aphid overwintering

Abstract.  1. When first instar nymphs and adults of the grain aphid Sitobion avenae (Fabricius) (Hemiptera: Aphidiae) were maintained in long-term cultures (>6 months) at 20 °C and 10 °C, the LT₅₀ decreased from −8 and −8.8 °C to −16.0 and −13.5 °C, respectively.
2. When aphids from the 20 °C culture were transferred to 10 °C, there was a progressive increase in cold tolerance through three successive generations. Transfer of newly moulted pre-reproductive adults reared at 10 °C for three generations back to 20 °C resulted in a rapid loss of cold hardiness in their nymphal offspring.
3. In all generations reared at 10 °C, first born nymphs were more cold hardy than those born later in the birth sequence. The LT₅₀ of nymphs produced on the first day of reproduction in the first, second and third generations maintained at 10 °C were −14.8, −17.0 and −16.6 °C, respectively. Thereafter, nymphal cold hardiness decreased over the subsequent 14 days of reproduction in each generation at 10 °C with mean LT₅₀ values of −10.3, −12.6 and −14.8 °C, respectively. By contrast, the cold tolerance of first born nymphs of aphids reared continuously at 20 °C did not differ in comparison with later born siblings. The LT₅₀ of adult aphids was also unaffected by ageing.
4. The ecological relevance of these findings is discussed in relation to the overwintering survival of aphids such as S. avenae .     

Leaf age as a risk factor in contamination of lettuce with Escherichia coli O157:H7 and Salmonella enterica

Outbreaks of Escherichia coli O157:H7 infections have been linked increasingly to leafy greens, particularly to lettuce. We present here the first evidence that this enteric pathogen can multiply on the leaves of romaine lettuce plants. The increases in population size of E. coli O157:H7 in the phyllosphere of young lettuce plants ranged from 16- to 100-fold under conditions of warm temperature and the presence of free water on the leaves and varied significantly with leaf age. The population size was consistently ca. 10-fold higher on the young (inner) leaves than on the middle leaves. The growth rates of Salmonella enterica and of the natural bacterial microflora were similarly leaf age dependent. Both enteric pathogens also achieved higher population sizes on young leaves than on middle leaves harvested from mature lettuce heads, suggesting that leaf age affects preharvest as well as postharvest colonization. Elemental analysis of the exudates collected from the surfaces of leaves of different ages revealed that young-leaf exudates were 2.9 and 1.5 times richer in total nitrogen and carbon, respectively, than middle-leaf exudates. This trend mirrored the nitrogen and carbon content of the leaf tissue. Application of ammonium nitrate, but not glucose, to middle leaves enhanced the growth of E. coli O157:H7 significantly, suggesting that low nitrogen limits its growth on these leaves. Our results indicate that leaf age and nitrogen content contribute to shaping the bacterial communities of preharvest and postharvest lettuce and that young lettuce leaves may be associated with a greater risk of contamination with E. coli O157:H7.