Variability in survival of Pectinatus cerevisiiphilus, strictly anaerobic bacteria, under different oxygen conditions

Survival of Pectinatus cerevisiiphilus DSM 20466 in pure culture at variable temperatures under different oxygen concentrations was measured. Survival of P. cerevisiiphilus in co-culture with Saccharomyces cerevisiae under both saturated oxygen and brewing conditions was also studied. The survival of strictly anaerobic bacteria to oxygen seems to follow the classical laws of heat resistance. The D(oxy) values of P. cerevisiiphilus , calculated as a function of oxygen level, shows that the oxygen level is important for the survival duration of the bacteria. The temperature greatly influences the oxygen resistance of P. cerevisiiphilus, which increases when the temperature decreases. P. cerevisiiphilus resists better in co-culture than in pure culture under saturated oxygen conditions. Therefore, the oxygenation of the wort does not totally eliminate the risk of beer contamination by this bacterium. Under brewing conditions in co-culture at 8 degrees C, P. cerevisiiphilus grows slowly to reach a final cell concentration up to 10(6) cells/mL in beer, which is undrinkable. Pectinatus is a strictly anaerobic bacterium; however, it is resistant under certain oxygen conditions of incubation. This resistance is considerably higher in the presence of Saccharomyces cerevisiae .     

Surface-associated, PrfA-regulated proteins of Listeria monocytogenes synthesized under stress conditions

The expression of the five clustered genes of Listeria monocytogenes: plcA, hly, mpl, actA and plcB is under the control of the positive regulation factor PrfA. Listeriolysin, encoded by the hly gene, is the only prominent PrfA-controlled gene product observed when L. monocytogenes strain NCTC 7973 is cultured in a rich medium at 37°C to the logarithmic growth phase. Stress conditions such as heat-shock or stationary culture conditions lead to the induction of additional PrfA-dependent proteins (PdPs): ActA (92 kDa), a 38kDa protein of unknown function and a 34kDa protein which probably represents PlcA. Under nutrient-stress conditions PdPs are preferentially synthesized and in addition to the already known PdPs at least five new, not yet functionally identified PdPs are detected. All PdPs are either secreted or are localized at the cell surface. Differences in the amount as well as the sizes of the PdPs are observed in different L. monocytogenes strains.     

Modelling the growth,survival and death of Listeria monocytogenes

In this paper, the predictive microbiology approach has been generalized to the study of growth, survival and death of Listeria monocytogenes. As this micro-organism is involved in food poisoning, its growth, survival and death were studied as functions of low temperatures, NaCl and phenol compounds, in a synthetic medium, by a factorially designed experiment. A significant inactivation of L. monocytogenes was obtained with 20 ppm of phenol and 4% (w/v) NaCl at temperatures from 4 to 12 degrees C. An empirical model is proposed to describe, in a single step, the biomass profile vs studied factors. Thereby, the influence of temperature, NaCl and phenol concentration on L. monocytogenes biomass quantity (0.5-8 log cfu ml(-1)) are presented as a function of storage duration. The comparisons of the proposed model with existing models (Gompertz for growth, vitalistic for survival and death) were performed. The use of a single equation allows the prediction of contamination levels in all experimental conditions without knowledge a priori. The model offers considerable prospects for its use in food microbiology.     

Listeria monocytogenes internalin and E-cadherin: from structure to pathogenesis

Many bacterial pathogens that invade non-phagocytic cells first interact with host cell surface receptors. Adhesion to the host cell is followed by the activation of specific host signalling pathways that mediate bacterial internalization. The food-borne Gram-positive bacterium Listeria monocytogenes makes use of two surface proteins, internalin (InlA) and InlB to engage in a species-specific manner the adhesion molecule E-cadherin and the hepatocyte growth factor receptor Met, respectively, to induce its internalization. After entry, Listeria has the capacity to spread from cell to cell and disseminate to its target organs after breaching the intestinal, blood–brain and placental barriers in human. InlA but not InlB is critical for the crossing of the intestinal barrier, whereas the conjugated action of both InlA and InlB mediates the crossing of the placental barrier. Here we review the InlA–E-cadherin interaction, the signalling downstream of this interaction, the molecular mechanisms involved in bacterial internalization and the role of InlA–E-cadherin interaction in the breaching of host barriers and the progression to listeriosis. Together, this review illustrates how in vitro data were validated by epidemiological approaches and in vivo studies using both natural hosts and genetically engineered animal models, thereby elucidating key issues of listeriosis pathophysiology.     

Effects of growth temperature and strictly anaerobic recovery on the survival of Listeria monocytogenes during pasteurization

Listeria monocytogenes F5069 was suspended in either Trypticase soy broth-0.6% yeast extract (TSBYE) or sterile, whole milk and heated at 62.8 degrees C in sealed thermal death time tubes. Severely heat-injured cells were recovered in TSBYE within sealed thermal death time tubes because of the formation of reduced conditions in the depths of the TSBYE. Also, the use of strictly anaerobic Hungate techniques significantly increased recovery in TSBYE containing 1.5% agar compared with aerobically incubated controls. The exogenous addition of catalase, but not superoxide dismutase, slightly increased the recovery of heat-injured cells in TSBYE containing 1.5% agar incubated aerobically. Growth of cells at 43 degrees C caused a greater increase in heat resistance as compared with cells heat shocked at 43 degrees C or cells grown at lower temperatures. Growth of L. monocytogenes at 43 degrees C and enumeration by the use of strictly anaerobic Hungate techniques resulted in D62.8 degrees C values that were at least sixfold greater than those previously obtained by using cells grown at 37 degrees C and aerobic plating. Results indicate that, under the conditions of the present study, high levels of L. monocytogenes would survive the minimum low-temperature, long-time treatment required by the U.S. Food and Drug Administration for pasteurizing milk. The possible survival of low levels of L. monocytogenes during high-temperature, short-time pasteurization and enumeration of injured cells by recovery on selective media under strictly anaerobic conditions are discussed.     

A tandem repeat of a fragment of Listeria monocytogenes internalin B protein induces cell survival and proliferation

Hepatocyte growth factor (HGF) is critical for tissue homeostasis and repair in many organs including the lung, heart, kidney, liver, nervous system, and skin. HGF is a heterodimeric protein containing 20 disulfide bonds distributed among an amino-terminal hairpin, four kringle domains, and a serine protease-like domain. Due to its complex structure, recombinant production of HGF in prokaryotes requires denaturation and refolding, processes that are impractical for large-scale manufacture. Thus, pharmaceutical quantities of HGF are not available despite its potential applications. A fragment of the Listeria monocytogenes internalin B protein from amino acids 36-321 (InlB??????) was demonstrated to bind to and partially activate the HGF receptor Met. InlB?????? has a stable β-sheet structure and is easily produced in its native conformation by Escherichia coli. We cloned InlB?????? (1×InlB??????) and engineered a head-to-tail repeat of InlB?????? with a linker peptide (2×InlB??????); 1×InlB?????? and 2×InlB?????? were purified from E. coli. Both 1× and 2×InlB?????? activated the Met tyrosine kinase. We subsequently compared signal transduction of the two proteins in primary lung endothelial cells. 2×InlB?????? activated ERK1/2, STAT3, and phosphatidylinositol 3-kinase/Akt pathways, whereas 1×InlB?????? activated only STAT3 and ERK1/2. The 2×InlB?????? promoted improved motility compared with 1×InlB?????? and additionally stimulated proliferation equivalent to full-length HGF. Both the 1× and 2×InlB?????? prevented apoptosis by the profibrotic peptide angiotensin II in cell culture and ex vivo lung slice cultures. The ease of large-scale production and capacity of 2×InlB?????? to mimic HGF make it a potential candidate as a pharmaceutical agent for tissue repair.     

Growth and reducing capacity of Listeria monocytogenes under different initial redox potential

Aims:  To determine the reducing capacity of Listeria monocytogenes and to highlight the effect of redox potential on its growth parameters.
Methods and Results:  The reducing capacity of L. monocytogenes was monitored in Brain Heart Infusion Broth media at different initial redox potential (Eh) and pH at 37°C. The effect of Eh obtained by gas flushing (air, N₂ and N₂-H₂) or by adding potassium ferricyanide and dithiotreitol in concentration from 1 to 10 mmol l⁻¹on L. monocytogenes growth parameters at pH 6·0, 7·0 and 8·0 was investigated. A total change of 539 mV (±44 mV) from an initial redox value of +330 ± 8 mV to a more negative potential in redox curves was observed resulting from L. monocytogenes growth at pH 7·0 at 37°C. A significant influence of pH and redox potential on L. monocytogenes lag phase of growth was shown ( P  < 0·05).
Conclusions:  Listeria monocytogenes exhibited longer lag phase in reducing conditions and at pH 6·0. The method used to modify the redox potential was shown to have no effect on growth parameters at pH 7·0.
Significance and Impact of the Study:  The provided information on the extending lag time and the possible delayed growth of this major pathogen in reducing conditions might be useful for its control in foods.     

Growth of desulfovibrio desulfuricans under heterotrophic and anaerobic conditions

Alico, Robert K. (St. Bonaventure University, St. Bonaventure, N.Y.), and Francis W. Liegey. Growth of Desulfovibrio desulfuricans, under heterotrophic and anaerobic conditions. J. Bacteriol. 91:1112-1114. 1966.-Growth of Desulfovibrio desulfuricans was investigated under heterotrophic and anaerobic conditions. For initial growth to occur, it was found that the E(h) or redox potential must be at least 0 mv. Some carbon sources were tested, and those which could be metabolized by D. desulfuricans were pyruvate, lactate, glycerol, glyceraldehyde, and ribose. Observations were also made on the sulfate used during growth. Various amounts of sulfate were added and depleted within 48 hr. This may be correlated with the decline in growth. As the terminal electron acceptor was exhausted the organisms could not respire, and, with the subsequent energy depletion, the population decreased.     

一多重耐药单增李斯特菌株的生长特性及毒力分析

为了解单增李斯特菌株耐药后可能发生的生物学变化,以哈市生肉中分离到的1株对17种抗生素耐受的单增李斯特菌株L.M.B8为研究对象,对其生长及毒力特性进行研究。结果显示,L.M.B8的生长及毒力特性均与标准菌株有明显差异。在NaC l浓度为0.5%~5%、pH值为4.0~10.0及温度为20~45℃范围内,L.M.B8的生长速度均明显高于标准菌株。L.M.B8对高浓度盐的敏感性高于标准菌株,且对温度的适应能力强于标准菌株。从生长曲线看,L.M.B8的对数生长期与稳定期均较标准菌株提前2~3 h,且其稳定期较标准菌株明显缩短。L.M.B8小鼠腹腔注射半数致死量（LD50）较标准菌株明显降低。该研究为进一步探讨单增李斯特菌的耐药性与其他生物学特性的相关性奠定基础。     

Effects of growth temperature and strictly anaerobic recovery on the survival of Listeria monocytogenes during pasteurization.

Listeria monocytogenes F5069 was suspended in either Trypticase soy broth-0.6% yeast extract (TSBYE) or sterile, whole milk and heated at 62.8 degrees C in sealed thermal death time tubes. Severely heat-injured cells were recovered in TSBYE within sealed thermal death time tubes because of the formation of reduced conditions in the depths of the TSBYE. Also, the use of strictly anaerobic Hungate techniques significantly increased recovery in TSBYE containing 1.5% agar compared with aerobically incubated controls. The exogenous addition of catalase, but not superoxide dismutase, slightly increased the recovery of heat-injured cells in TSBYE containing 1.5% agar incubated aerobically. Growth of cells at 43 degrees C caused a greater increase in heat resistance as compared with cells heat shocked at 43 degrees C or cells grown at lower temperatures. Growth of L. monocytogenes at 43 degrees C and enumeration by the use of strictly anaerobic Hungate techniques resulted in D62.8 degrees C values that were at least sixfold greater than those previously obtained by using cells grown at 37 degrees C and aerobic plating. Results indicate that, under the conditions of the present study, high levels of L. monocytogenes would survive the minimum low-temperature, long-time treatment required by the U.S. Food and Drug Administration for pasteurizing milk. The possible survival of low levels of L. monocytogenes during high-temperature, short-time pasteurization and enumeration of injured cells by recovery on selective media under strictly anaerobic conditions are discussed.     

Sources and survival of Listeria monocytogenes on fresh,leafy produce

Listeria monocytogenes is an intracellular human pathogen which enters the body through contaminated food stuffs and is known to contaminate fresh leafy produce such as spinach, lettuce and rocket. Routinely, fresh leafy produce is grown and processed on a large scale before reaching the consumer through various products such as sandwiches and prepared salads. From farm to fork, the fresh leafy produce supply chain (FLPSC) is complex and contains a diverse range of environments where L. monocytogenes is sporadically detected during routine sampling of produce and processing areas. This review describes sources of the bacteria in the FLPSC and outlines the physiological and molecular mechanisms behind its survival in the different environments associated with growing and processing fresh produce. Finally, current methods of source tracking the bacteria in the context of the food supply chain are discussed with emphasis on how these methods can provide additional, valuable information on the risk that L. monocytogenes isolates pose to the consumer.     

Methods for the isolation and identification of Listeria spp. and Listeria monocytogenes: a review

Listeria monocytogenes is an important food-borne pathogen and is widely tested for in food, environmental and clinical samples. Identification traditionally involved culture methods based on selective enrichment and plating followed by the characterization of Listeria spp. based on colony morphology, sugar fermentation and haemolytic properties. These methods are the gold standard; but they are lengthy and may not be suitable for testing of foods with short shelf lives. As a result more rapid tests were developed based on antibodies (ELISA) or molecular techniques (PCR or DNA hybridization). While these tests possess equal sensitivity, they are rapid and allow testing to be completed within 48 h. More recently, molecular methods were developed that target RNA rather than DNA, such as RT-PCR, real time PCR or nucleic acid based sequence amplification (NASBA). These tests not only provide a measure of cell viability but they can also be used for quantitative analysis. In addition, a variety of tests are available for sub-species characterization, which are particularly useful in epidemiological investigations. Early typing methods differentiated isolates based on phenotypic markers, such as multilocus enzyme electrophoresis, phage typing and serotyping. These phenotypic typing methods are being replaced by molecular tests, which reflect genetic relationships between isolates and are more accurate. These new methods are currently mainly used in research but their considerable potential for routine testing in the future cannot be overlooked.     

Methods of assessing microbial activity and inhibition under anaerobic conditions: a literature review

This work reviews the existing methodologies for assessing microbial activity and inhibition under anaerobic conditions. The anaerobic digestion process consists of several metabolic steps–the Anaerobic Digestion Model No. 1 (ADM1) has attempted to describe these steps in the form of a mathematical model with the intention of providing a reference base for all further efforts in the modelling of anaerobic processes. The existence of a reference point for modelling has highlighted the fact that there is a lack of coherence between the many different methodologies for experimentally assessing anaerobic activity and inhibition.A working group of the International Water Association was recently founded to harmonise the existing methodologies with the ultimate intention of developing a unified reference procedure– a primary objective of the group will be the establishment of a standard terminology in the field of anaerobic digestion, activity and inhibition assessment. Secondly, it will compare the existing methodologies and develop standard protocols for assessing the kinetic parameters (e.g. maximum uptake rate, half-saturation constant) of anaerobic processes that may be entered directly into ADM1 and its successors.This paper revises and enlarges a contribution presented by the authors at the workshop Harmonisation of anaerobic biodegradation, activity and inhibition assays (Ligthart & Nieman 2002, Proc. workshop held in Orta (Italy) June 7–8, 2002) and aims to promote a clear understanding of the currently established methodology.Numerous methods have been developed over the past 30 years, since Van den Berg et al. (1974, Biotechnol Bioeng 16(11)– 1459–1469) measured methanogenic activity, by using a manometric device equipped with a photoelectric sensor to quantify the gas production. Methanogenesis is often the rate limiting step of the entire process and since the quantification of gas flowrate is relatively easy to perform, most of the methods reported in literature monitor the production of biogas. These methods can be termed volumetric or manometric methods, as the volume of biogas produced or the pressure increase due to gas production inside a close vessel are assessed, respectively. However, this same concept can be employed to assess activity or inhibition of individual metabolic steps preceding the methanogenic one, providing that they are rate limiting for the whole process. The reliability of activity assessment through gas measurement has been proven to be strongly dependent on the equilibrium between liquid and gas phase in a closed vessel. This can be influenced by many factors, e.g. the amount and characteristics of the test substrate; the concentration of the biomass; the gas-to-liquid ratio– all these aspects will need to be addressed in the standard procedure. Other direct or indirect methods, targeting physico-chemical or microbiological parameter exist and have been investigated by many authors. Besides the interest for research purposes, the definition of reference methods to assess activity and inhibition can be of great interest for engineers, both phy. Specific reference procedures might be needed for particular applications, e.g. the (kinetic) study of rate limiting microbial steps and might require ad-hoc methodologies to be devised. A microbiological technique such as FISH, coupled with microsensors have been reported to have a great potential in the near future.Passed away on April 7th 2003.     

Vertical migration, nitrate uptake and denitrification: survival mechanisms of foraminifers (Globobulimina turgida) under low oxygen conditions

(15)NO(3)(-) isotope labelling experiments were performed to investigate foraminiferal nitrate uptake strategies and the role of pseudopodial networks in nitrate uptake. Globobulimina turgida were placed below the nitrate penetration depth in homogenized sediment cores incubated in artificial seawater containing (15)NO(3)(-) . A nylon net prevented the vertical migration of foraminifera to strata containing nitrate and oxygen, but allowed potential access to such strata by extension of pseudopods. No (15)NO(3)(-) was found in G. turgida in these cores, suggesting that foraminifera cannot extend their pseudopods for nitrate uptake through several millimetres of sediment, but must physically migrate upwards closer to nitrate-containing strata. However, foraminiferal migration patterns in control cores with no nylon net were erratic, suggesting that individuals move in random orientations until they find favourable conditions (i.e. free nitrate or oxygen). A second experiment showed that foraminifera actively collect nitrate both in the presence and in the absence of oxygen, although uptake was initiated faster if oxygen was absent from the environment. However, no systematic influence of the size of the intracellular nitrate pool on nitrate uptake was observed, as specimens containing a large range of intracellular nitrate (636-19 992 pmol per cell) were measured to take up (15)NO(3)(-) at comparable rates.     

Growth rate and growth probability of Listeria monocytogenes in dairy, meat and seafood products in suboptimal conditions

AIMS: To evaluate the performances of models predicting the growth rate or the growth probability of Listeria monocytogenes in food. METHODS AND RESULTS: Cardinal and square root type models including or not interactions between environmental factors and probability models were evaluated for their ability to describe the behaviour of L. monocytogenes in liquid dairy products, cheese, meat and seafood products. Models excluding interactions seemed sufficient to predict the growth rate of L. monocytogenes. However, the accurate prediction of growth/no-growth limits needed to take interactions into account. A complete and a simplified form (preservatives deducted) of a new cardinal model including interactions and parameter values were suggested to predict confidence limits for the growth rate of L. monocytogenes in food. This model could also be used for the growth probability prediction. CONCLUSIONS: The new cardinal model including interactions was efficient to predict confidence limits for the growth rate of L. monocytogenes and its growth probability in liquid dairy products, meat and seafood products. In cheese, the model was efficient to predict the absence of growth of the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The suggested model can be used for risk assessment and risk management concerning L. monocytogenes in dairy, meat and seafood products.     

Growth inhibition of Listeria monocytogenes by a nonbacteriocinogenic Carnobacterium piscicola

AIMS: This study elucidates the mechanisms by which a nonbacteriocinogenic Carnobacterium piscicola inhibits growth of Listeria monocytogenes. METHODS AND RESULTS: Listeria monocytogenes was exposed to live cultures of a bacteriocin-negative variant of C. piscicola A9b in co-culture, in a diffusion chamber system, and to a cell-free supernatant. Suppression of maximum cell density (0-3.5 log units) of L. monocytogenes was proportional to initial levels of C. pisciola (10(3)-10(7) CFU ml(-1)). Cell-to-cell contact was not required to cause inhibition. The cell-free C. piscicola supernatant caused a decrease in L. monocytogenes maximum cell density, which was abolished by glucose addition but not by amino acid, vitamin or mineral addition. The fermentate also gave rise to a longer lag phase and a reduction in growth rate. These effects were independent of glucose and may have been caused by acetate production by C. piscicola. 2D gel-electrophoretic patterns of L. monocytogenes exposed to C. piscicola or to L. monocytogenes fermentate did not differ. Treatment with C. piscicola fermentate resulted in down-regulation (twofold) of genes involved in purine- or pyrimidine metabolism, and up-regulation (twofold) of genes from the regulon for vitamin B12 biosynthesis and propanediol and ethanolamine utilization. CONCLUSIONS: A nonbacteriocinogenic C. piscicola reduced growth of L. monocytogenes partly by glucose depletion. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mechanism of microbial interaction enhances prediction of growth in mixed communities as well as use of bioprotective principles for food preservation.