Development of an improved chemically defined minimal medium for Listeria monocytogenes.

A chemically defined minimal medium for Listeria monocytogenes has been developed by modification of Welshimer's medium. The growth factors required by L. monocytogenes Scott A are leucine, isoleucine, arginine, methionine, valine, cysteine (each at 100 mg/liter), riboflavin and biotin (each at 0.5 micrograms/ml), thiamine (1.0 micrograms/ml), and thioctic acid (0.005 micrograms/ml). Growth was stimulated by 20 micrograms of Fe3+ per ml as ferric citrate. Glucose (1%) and glutamine (600 mg/liter) are required as primary sources of carbon and nitrogen. Glucose could not be replaced by various organic acids or amino acids. Of several sugars tested, fructose, mannose, cellobiose, trehalose, maltose (weak), glycerol (weak), and the amino sugars glucosamine, N-acetylglucosamine, and N-acetylmuramic acid supported growth in the absence of glucose. Evidence was found that chitin and cell walls of starter bacteria (Lactococcus lactis) supported survival of L. monocytogenes, which suggests that the pathogen may obtain carbon and energy sources during colonization of some foods, such as cheeses, by assimilating bacteria or molds that are present.     

Aerobic and anaerobic metabolism in Entamoeba histolytica

Respiration by Entamoeba histolylica is confirmed. A doubling of the rate of oxygen uptake was observed upon the addition of d-glucose to cells in which the glycogen reserve had been partially depleted. In cells metabolizing endogenous substrates the rate of oxygen uptake was not influenced by sodium cyanide or sodium succinate. It was slightly depressed when d-mannose was the added sugar. The end products, CO₂, ethanol, and acetate accounted for essentially all of the glucose carbon utilized in both aerobic and anaerobic experiments. The radioactivity from uniformly labelled ¹⁴C-glucose was found in these products. Three times as much ethanol as acetate was produced in the anaerobic experiments and in the aerobic experiments this ratio was approximately reversed.     

Aerobic and anaerobic metabolism during activity in small rodents.

Analysis of oxygen consumption and lactic acid formation during five minutes of maximal activity by the rodents Microtus montanus (Cricetidae) and Dipodomys merriami (Hetermyidae) indicates that: (1) anaerobiosis provides approximately 10% of total energy utilized during the 5-minute activity period; (2) anaerobiosis may account for as much as one-third of total energy utilized during the first 30 seconds of activity. In addition, these data indicate at least one species of lizard may be capable of a higher total rate of metabolism during "burst" activity than are the rodents investigated here.     

Role of the glycine betaine and carnitine transporters in adaptation of Listeria monocytogenes to chill stress in defined medium

The food-borne pathogen Listeria monocytogenes proliferates at refrigeration temperatures, rendering refrigeration ineffective in the preservation of Listeria-contaminated foods. The uptake and intracellular accumulation of the potent compatible solutes glycine betaine and carnitine has been shown to be a key mediator of the pathogen's cold-tolerant phenotype. To date, three compatible solute systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and the carnitine transporter OpuC. We investigated the specificity of each transporter towards each compatible solute at 4 degrees C by examining mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state compatible solute accumulation data together with growth rate experiments demonstrated that under cold stress glycine betaine transport is primarily mediated by Gbu and that Gbu-mediated betaine uptake results in significant growth stimulation of chill-stressed cells. BetL and OpuC can serve as minor porters for the uptake of betaine, and their action is capable of providing a small degree of cryotolerance. Under cold stress, carnitine transport occurs primarily through OpuC and results in a high level of cryoprotection. Weak carnitine transport occurs via Gbu and BetL, conferring correspondingly weak cryoprotection. No other transporter in L. monocytogenes 10403S appears to be involved in transport of either compatible solute at 4 degrees C, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown at that temperature.     

Aerobic and anaerobic metabolism during activity in snakes

Summary Oxygen consumption and blood lactate concentration in the snakesColuber constrictor, Crotalus viridis, Lichanura roseofusca andMasticophis flagellum and whole body lactate concentration inCrotalus viridis andMasticophis flagellum were determined under standard conditions and after a bout of maximal activity induced by a 5 min period of mechanical stimulation. Observations were made atT _b=35°C inColuber, Crotalus, andMasticophis and 32°C inLichanura. Maximal oxygen consumption inColuber andMasticophis was twice that ofCrotalus and 4 x that ofLichanura (Fig. 1). Post-active whole body lactate concentration inMasticophis was twice that ofCrotalus (Fig. 2). Immediately post-active and 30 min post-active blood lactate concentration inColuber andMasticophis was 1.5 x and 3.5 x that ofCrotalus andLichanura, respectively (Fig. 3). These data support conclusions that: (a) maximal energy production by these snakes correlates well with their respective modes of predation and defense, the highly active predatorsColuber andMasticophis being capable of the greatest net energy production during activity; (b)Coluber andMasticophis exhibit aerobic scopes as high or higher than any other comparably sized reptile heretofore investigated; (c) weight specific anaerobic metabolism probably does not decrease with increasing body size in reptiles; (d) anaerobic metabolism provides >50% of net energy production during five minutes of activity in all species examined (Table 1).     

Listeria monocytogenes Scott A transports glucose by high-affinity and low-affinity glucose transport systems.

Listeria monocytogenes transported glucose by a high-affinity phosphoenolpyruvate-dependent phosphotransferase system and a low-affinity proton motive force-mediated system. The low-affinity system (Km = 2.9 mM) was inhibited by 2-deoxyglucose and 6-deoxyglucose, whereas the high-affinity system (Km = 0.11 mM) was inhibited by 2-deoxyglucose and mannose but not 6-deoxyglucose. Cells and vesicles artificially energized with valinomycin transported glucose or 2-deoxyglucose at rates greater than those of de-energized cells, indicating that a membrane potential could drive uptake by the low-affinity system.     

Selective plating medium for quantitative recovery of food-borne Listeria monocytogenes.

A new plating medium (lithium chloride-ceftazidime agar [LCA]) was designed to quantitatively recover food-borne Listeria monocytogenes in the form of large colonies while inhibiting most other food-borne microorganisms. This medium included brain heart infusion agar as the nutritive agar base and a combination of selective agents (LiCl, glycine anhydride, and ceftazidime). Comparison of LCA and lithium chloride-phenylethanol-moxalactam agar (LPM) indicated that both were equally effective for the enumeration of the cold-tolerant pathogen in artificially and naturally contaminated foods. However, LCA was more effective than LPM in the recovery of sublethally heat-injured cells. Moreover, Listeria colonies on LCA exhibited a more distinct bluish hue than those on LPM when viewed by the Henry oblique transillumination technique.     

Aerobic and anaerobic metabolism of the freshwater oligochaete Tubifex sp.

The freshwater oligochaete Tubifex shows several mechanisms of metabolic adaptations, enabling the worms to occupy saprobial habitats of extremely variable oxygen content. Under normoxic conditions the metabolism of the worms is mainly aerobic with a respiratory ratio of 0.7. Under hypoxic conditions, metabolism of energy sources via aerobic and anaerobic pathways is observed. During complete anoxia acetate and propionate are the main products of glycogen degradation and they are excreted in constant rates into the water. A retransfer of the worms to aerobic conditions enables them to regain aerobic metabolic state within about 60 min.In two Tubifex habitats, which we have characterized, concentrations of dissolved organic material (DOM) were low in the surface water, but high in the interstitial water from sediments. The short-chain fatty acids acetate and propionate reached concentrations up to 1 mmole/liter. Employing radioisotope techniques, we demonstrated that Tubifex can achieve an integumentary uptake of acetate and propionate from artificial tap water at naturally occurring concentrations of 5 to 1000 M. Levels of uptake (600 to 800 nmoles/g wwt.hr) and transport characteristics are very similar to those of marine invertebrates associated with detritus rich sediments. The uptake is susceptible to inhibition by structurally analogous compounds and to metabolic inhibition. Furthermore, DOM uptake in Tubifex is susceptible to inhibition by oxygen depletion, ouabain and Na⁺-depletion. The results may suggest that a carrier system for DOM transport exists in the integument of the worms. The uptake system is highly specific for aliphatic C2 and C3 carboxylic acids. The absorbed volatile fatty acids are rapidly metabolized. Only 15 min after absorption, a considerable amount of radioactivity is present in the glycogen storage of the animals. Depending on the substrate concentration assumed to be available for uptake, up to 40 per cent of the oxidative requirement of the worms may be attained by using dissolved organic material from the interstitial water of their habitat.Supported by the Deutsche Forschungsgemeinschaft (Ho 631/9-9).     

Highly selective medium for isolation of Listeria monocytogenes from food.

A new selective medium (Al-Zoreky-Sandine listeria medium [ASLM]) was formulated to recover Listeria monocytogenes from food specimens; the medium completely inhibited common food microflora. Recognition of Listeria colonies is evident by black discoloration of the medium due to esculin hydrolysis without need for special illuminating equipment. The medium contains acriflavin, ceftazidime, and moxalactam as selective agents. Compared with Listeria Selective Agar, ASLM was equally effective in recovering L. monocytogenes. However, ASLM inhibited micrococci, enterococci, and gram-negative bacteria, especially a strain that mimicked L. monocytogenes on Listeria Selective Agar. The new medium was able to recover heat injured cells with only 15% less count than the nonselective medium.     

Temperature-Dependent Requirement for Catalase in Aerobic Growth of Listeria monocytogenes F2365

Listeria monocytogenes is a Gram-positive, psychrotrophic, facultative intracellular food-borne pathogen responsible for severe illness (listeriosis). The bacteria can grow in a wide range of temperatures (1 to 45°C), and low-temperature growth contributes to the food safety hazards associated with contamination of ready-to-eat foods with this pathogen. To assess the impact of oxidative stress responses on the ability of L. monocytogenes to grow at low temperatures and to tolerate repeated freeze-thaw stress (cryotolerance), we generated and characterized a catalase-deficient mutant of L. monocytogenes F2365 harboring a mariner-based transposon insertion in the catalase gene (kat). When grown aerobically on blood-free solid medium, the kat mutant exhibited impaired growth, with the extent of impairment increasing with decreasing temperature, and no growth was detected at 4°C. Aerobic growth in liquid was impaired at 4°C, especially under aeration, but not at higher temperatures (10, 25, or 37°C). Genetic complementation of the mutant with the intact kat restored normal growth, confirming that inactivation of this gene was responsible for the growth impairment. In spite of the expected impact of oxidative stress responses on cryotolerance, cryotolerance of the kat mutant was not affected.Listeria monocytogenes is a Gram-positive, facultative intracellular food-borne pathogen that has the ability to cause a severe disease (listeriosis) in humans and animals (13, 28, 30). L. monocytogenes is ubiquitously distributed in the environment and has the ability to grow over a wide range of temperatures (between 1 and 45°C) (13). Growth at low temperature has important implications for environmental persistence of the organism and for contamination of cold-stored, ready-to-eat foods, thus contributing to the food safety hazards associated with L. monocytogenes (19).L. monocytogenes is subjected to oxidative stress during both extracellular and intracellular growth and has evolved several responses to minimize the impact of reactive oxygen species (ROS). Catalase and superoxide dismutase (SOD) work synergistically in detoxification of ROS: superoxide anions are converted to H₂O₂ by SOD, with subsequent conversion of H₂O₂ into water and oxygen by catalase (22). Exposure to ROS may be especially acute during intracellular infection as well as under certain environmental conditions, such as those involved in repeated freezing and thawing (15, 16, 23, 29, 33).Previous studies revealed that the ability of L. monocytogenes to survive repeated freezing and thawing (cryotolerance) was markedly dependent on growth temperature, with bacteria grown at 37°C having significantly higher cryotolerance than those grown at either 4 or 25°C (1). However, mechanisms underlying Listeria''s cryotolerance have not been identified. Since oxidative damage is considered to take place during freezing and thawing, determinants such as catalase may be involved in cryotolerance.The catalase of L. monocytogenes has been investigated primarily in terms of its potential role in pathogenesis, with somewhat conflicting results. The isolation of catalase-negative strains from human listeriosis patients has led to the speculation that catalase is not required for human virulence (4, 8, 12, 31). On the other hand, under certain conditions (e.g., reduced serum levels), catalase-negative strains were impaired in their ability to survive in activated macrophages in comparison to catalase-positive strains (32). Furthermore, the catalase gene kat was among those for which expression was induced in infected cell cultures and in the spleens of mice infected with L. monocytogenes EGD-e, suggesting possible contributions to pathogenesis (5, 9).The potential role of catalase in environmental adaptations of L. monocytogenes such as growth at low temperature and cryotolerance was not addressed in these earlier investigations. In this study, we have characterized an isogenic mutant of L. monocytogenes F2365 to determine the involvement of catalase in growth at different temperatures, survival in selected foods, and cryotolerance of L. monocytogenes.     

Plasmids in Listeria monocytogenes and other Listeria species.

One hundred and twenty-two food, clinical, and veterinary strains of Listeria monocytogenes were examined for the presence of plasmids. Twenty-five (20%) contained plasmids, which varied from 1.3 to 66 MDa in size. Of 10 strains of other Listeria species (L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, L. grayi, and L. murrayi) examined, seven (70%) contained plasmids, varying from 38 to 53 MDa. No strains with multiple plasmids were found. Plasmids of identical size were isolated from related strains in some, although not all, cases. The presence of a plasmid in a strain was not related to phenotypic characters of known extrachromosomal inheritance.     

Development of a synthetic minimal medium for Listeria monocytogenes

A defined solid and liquid minimal medium, HTM, which contained methionine and cysteine as the sole amino acids, was developed for Listeria monocytogenes. Complex broth-grown L. monocytogenes had to adapt to HTM by inducing amino acid biosyntheis. HTM is the simplest minimal medium available for growth of L. monocytogenes.     

Carbon metabolism of Listeria monocytogenes growing inside macrophages

The intracellular metabolism of Listeria monocytogenes was studied by ¹³C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-¹³C₆]glucose prior to infection, but not during infection, label was detected only in Ala, Asp and Glu of the macrophage and bacterial protein with equal isotope distribution. When [U-¹³C₆]glucose was provided during the infection period, ¹³C label was found again in Ala, Asp and Glu from host and bacterial protein, but also in Ser, Gly, Thr and Val from the bacterial fraction. Mutants of L. monocytogenes defective in the uptake and catabolism of the C₃-metabolites, glycerol and/or dihydroxyacetone, showed reduced incorporation of [U-¹³C₆]glucose into bacterial amino acids under the same experimental settings. The ¹³C pattern suggests that (i) significant fractions (50–100%) of bacterial amino acids were provided by the host cell, (ii) a C₃-metabolite can serve as carbon source for L. monocytogenes under intracellular conditions and (iii) bacterial biosynthesis of Asp, Thr and Glu proceeds via oxaloacetate by carboxylation of pyruvate.     

Bacteriocin inhibition of two glucose transport systems in Listeria monocytogenes

Listeria monocytogenes transports glucose by proton motive force-mediated and phosphoenolpyruvate-dependent phosphotransferase systems (PEP-dependent PTS). Inhibition of both systems by nisin, pediocin JD and leuconosin S is reported here for four strains of L. monocytogenes . Intracellular and extracellular adenosine triphosphate (ATP) and extracellular inorganic phosphate were measured in energized L. monocytogenes Scott A cells to determine whether inhibition of the PEP-dependent PTS might occur as a result of bacteriocin-induced leakage of intracellular components. Addition of nisin resulted in a decrease in intracellular ATP with an increase in extracellular ATP. Leuconosin S and pediocin JD induced a depletion of intracellular ATP. ATP efflux was low for the leuconosin S-treated cells and barely detectable for pediocin JD-treated cells. Addition of nisin, leuconosin S and pediocin JD induced efflux of inorganic phosphate. It appears that bacteriocin-mediated inhibition of the glucose PEP-dependent PTS occurs as a result of hydrolysis or efflux of ATP, PEP and other essential molecules from L. monocytogenes cells.     

Aerobic versus anaerobic metabolism of halogenated anilines by aParacoccus sp.

AParacoccus sp. which transforms aniline and different halogen-substituted derivatives under aerobic and anaerobic conditions was isolated from the soil. In experiments with¹⁴C-ring-labeled 4-chloroaniline, approximately 60% of the radioactive material disappeared from the growth medium after incubation under anaerobiosis within 48 hr, but under aerobic conditions no decrease of radioactivity in the growth medium was observed, although 4-chloroaniline was completely metabolized. Acetylation appears to constitute, especially under aerobic conditions, a major transformation mechanism by the bacterium, since almost 50% of the acetylated compound could be detected and identified if aniline, 2-, 3-, and 4-chloroaniline served as substrate. The formation of different metabolites under aerobic and anaerobic conditions clearly indicates the existence of two separate pathways in the metabolism of aniline compounds depending on the oxygen status of the environment.     

Aerobic and anaerobic carbohydrate metabolism and egg production of Schistosoma mansoni in vitro.

When S. mansoni adults were cultured in vitro for 12 days in a diphasic medium, their gross morphology, motor activity, frequency of sexual pairings, rates of glucose utilization and of lactic acid production were the same in the presence (90% N2/5% O2/5% CO2) or absence (95% N2/5% CO2) of oxygen. Therefore, no Pasteur effect, nor any reduction in lactic acid formation, was demonstrable under aerobic conditions. While aerobic conditions did not affect the rate of glycolysis, they had a marked effect on egg production. In the presence of oxygen, the rate of egg-laying reached a maximum between days 4 and 6. The average number of viable eggs produced per worm pair during this period was 118 (Sx equals 2.2), which is within the overall range (68 to 248) recorded by others for this same strain in vivo. Conversely, under anaerobic conditions in vitro, virtually no eggs were laid. It remains to be determined whether oxidative metabolism actually is required for energy to produce eggs, or whether some reaction yielding no ATP is essential for completion of their developmental process, such as tanning of the eggshall brought about by the oxidation of some phenolic compounds.     

Influence of interactions between temperature,ferric ammonium citrate and glycine betaine on the growth of Listeria monocytogenes in a defined medium

AIMS: To investigate interactions, if any, between temperature, ferric ammonium citrate and glycine betaine on the growth of Listeria monocytogenes in modified Pine's medium (Pine et al. 1989). METHODS AND RESULTS: Modified Pine's medium containing 0, 0.044, 0.088 or 0.176 g l(-1) ferric ammonium citrate, and 0 or 1 mM glycine betaine, was inoculated with each of two L. monocytogenes strains and incubated at 4, 25 or 37 degrees C. The optical density at 600 nm, and cell numbers, were determined at appropriate time intervals. At 4 degrees C, but not other temperatures, increasing ferric ammonium citrate resulted in improved growth in the absence, but not the presence, of glycine betaine. The presence of glycine betaine was inhibitory at 25 and 37 degrees C, but not at 4 degrees C. CONCLUSIONS: Interactions affecting the growth kinetics of L. monocytogenes were apparent between the parameters investigated. SIGNIFICANCE AND IMPACT OF THE STUDY: Limitations on the use of modified Pine's medium, and the significance of iron metabolism at lower temperatures, were revealed.