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T Abee

Publications and source records attributed to T Abee.

At least 19 recordsLinked to original sources
PubMed2005

Acid resistance variability among isolates of Salmonella enterica serovar Typhimurium DT104.

AIMS: Acid resistance could be an indicator of virulence since acid resistant strains are able to better survive the human stomach passage and in macrophages. We studied the acid resistance of several Salmonella Typhimurium DT104 strains isolated from food and humans and identified cellular parameters contributing to the enhanced acid resistance of these isolates. METHODS AND RESULTS: Acid resistance was tested in 37 Salmonella enterica Typhimurium serovar DT104 (S. Typhimurium DT104) strains. Acid adaptation at pH 5 followed by exposure for 2 h at pH 2.5 in the 27 human, nine nonhuman, and in two reference strains, revealed strong variation of acid survival. After 2 h at pH 2.5 six strains of S. Typhimurium DT104 were considered high acid resistant as they displayed a level of survival >10%, 14 strains were considered intermediate acid resistant (level of survival was <10% and >0.01%) and 19 strains were considered low acid resistant (level of survival <0.01%). Six strains were selected for further studies and proteomics revealed a relatively high amount of phase 2 flagellin in an acid-sensitive strain and a relatively high amount of the beta component of the H(+)/ATPase in an acid-resistant strain. Two strains were slightly more heat resistant possibly as the result of increased levels of DnaK or GroEL. CONCLUSIONS: A significant difference could be detected between human and food isolates regarding their acid resistance; all high acid-resistant strains were human isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: S. Typhimurium DT104 is known for two decades and has a great impact on human health causing serious food-borne diseases. Our results suggest the existence of a positive correlation between acid resistance and pathogenicity in S. Typhimurium DT104 as all high acid-resistant strains were isolated from humans.

Acids↗
PubMed2003

An antifungal compound produced by Bacillus subtilis YM 10-20 inhibits germination of Penicillium roqueforti conidiospores.

AIMS: To identify and characterize an antifungal compound produced by Bacillus subtilis YM 10-20 which prevents spore germination of Penicillium roqueforti. METHODS AND RESULTS: The antifungal compound was isolated by acid precipitation with HCl. This compound inhibited fungal germination and growth. Identification by HPLC and mass spectrometry analysis showed high similarity to iturin A. Permeabilization and morphological changes in P. roqueforti conidia in the presence of the inhibitor were revealed by fluorescence staining and SEM, respectively. CONCLUSOINS: The iturin-like compound produced by B. subtilis YM 10-20 permeabilizes fungal spores and blocks germination. SIGNIFICANCE AND IMPACT OF THE STUDY: Fluorescence staining in combination with flow cytometry and scanning electron microscopy are efficient tools for assessing the action of antifungal compounds against spores. Iturin-like compounds may permeabilize fungal spores and inhibit their germination.

Antifungal Agents↗
PubMed2003

Selection of fluorescent probes for flow cytometric viability assessment of Listeria monocytogenes exposed to membrane-active and oxidizing disinfectants.

The aim of this study was to select fluorescence methods for use as alternatives to plate counting to assess the viability of Listeria monocytogenes cells exposed to benzalkonium chloride (BAC) and hydrogen peroxide, two disinfectants with different mechanisms of action. A further aim of this study was to determine whether growth phase influences fluorescence labeling and whether it is possible to predict whether a probe will be a good viability indicator for cells exposed to a certain disinfectant on the basis of the mechanism of action of the disinfectant and the target of the fluorescent probe. The fluorescence methods used were labeling with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC; dehydrogenase activity), labeling with TOTO-1 iodide (TOTO; membrane-impermeant probe), and assessment of pH gradient maintenance in a low-pH buffer after labeling with the pH-sensitive probe 5-(and 6)-carboxyfluorescein succinimidyl ester (CFSE) (the pH(in) method). Growth phase influenced fluorescent labeling. However, the cutoff value for distinction between viable and nonviable cells was the same for both growth phases. The viability (determined by plate counts) of BAC-exposed cells correlated well with CTC labeling and TOTO exclusion. For both BAC-exposed and hydrogen peroxide-exposed cells, the pH(in) method gave a good qualitative indication of viability, sublethal damage, and cell death. CTC labeling and TOTO exclusion did not correlate with the viability of hydrogen peroxide-exposed cells. Our results demonstrate that even if the mechanism of action of a disinfectant is known, in some cases it is still difficult to predict whether a certain fluorescent probe is suitable for viability assessment. Thus, the proper selection of fluorescent probes for the assessment of the efficacy of antimicrobial agents is essential.

Benzalkonium Compounds↗
PubMed2002

The effect of the growth phase of Staphylococcus aureus on resistance to disinfectants in a suspension test.

The influence of growth phase on the resistance of Staphylococcus aureus to the surface-active agents benzalkonium chloride and dodecylbenzyl sulfonic acid and the oxidizing agents sodium hypochlorite and hydrogen peroxide was studied. The resistances of cells in different growth phases were compared to those of solid medium cells grown according to the European phase I suspension test. Using cells from different growth phases (+/- 3 x 10(7) CFU ml(-1)), we found that decline-phase cells were the most resistant cells. However, the decline-phase cell suspension contained more than 90% dead cells. A 10-fold-diluted suspension with a total concentration of cells equal to that of the other cell suspensions still revealed decline-phase cells to be generally the most resistant cell type. However, the resistance was drastically reduced, indicating that the large proportion of dead cells provided significant protection to the viable decline-phase cells. Hydrogen peroxide resistance could be partly explained by the high catalase activity in the dead-cell fraction. Exponential-phase cells were less resistant than decline-phase cells, and, surprisingly, stationary-phase cells were the least resistant of the three. Cells grown according to the European phase 1 suspension test were never the most resistant cells. Their survival was 1 to 3 log units lower than that of the most resistant cells. These findings show that the solid-medium cells currently used in disinfectant tests are not the most resistant cells that can be used.

Catalase↗
PubMed2001

Physiological function of exopolysaccharides produced by Lactococcus lactis.

The physiological function of EPS produced by Lactococcus lactis was studied by comparing the tolerance of the non-EPS producing strain L. lactis ssp. cremoris MG1614 and an EPS producing isogenic variant of this strain to several anti-microbial factors. There was no difference in the sensitivity of the strains to increased temperatures, freezing or freeze-drying and the antibiotics, penicillin and vancomycin. A model system showed that EPS production did not affect the survival of L. lactis during passage through the gastrointestinal tract although the EPS itself was not degraded during this passage. The presence of cell associated EPS and EPS in suspension resulted in an increased tolerance to copper and nisin. Furthermore, cell associated EPS also protected the bacteria against bacteriophages and the cell wall degrading enzyme lysozyme. However, it has not been possible, so far, to increase EPS production using the presence of copper, nisin, lysozyme or bacteriophages as inducing factors.

Bacteriophages↗
PubMed2001

DNA isolation protocols affect the detection limit of PCR approaches of bacteria in samples from the human gastrointestinal tract.

A major concern in molecular ecological studies is the lysis efficiency of different bacteria in a complex ecosystem. We used a PCR-based 16S rDNA approach to determine the effect of two DNA isolation protocols (i.e. the bead beating and Triton-X100 method) on the detection limit of seven feces-associated bacterial species of different genera. Glycogen was used in these protocols to improve the precipitation of small concentrations of DNA in ethanol without affecting the sequential procedures. The PCR detection limit of 16S rDNA amplicons on agarose gel from the seven strains tested varied between 8.0 (+/- 1.3) x 10(4) and 4.3 (+/- 1.6) x 10(6) cells for the bead beating method, and between 8.0 (+/- 1.3) x 10(4) and 5.4 (+/- 0.7) x 10(8) cells for the Triton X-100 method. These large differences are most like due to the difference in cell lysis efficiency, since a competitive PCR experiment did not indicate any preference for gram negative, low G+C gram positive or high G+C gram positive bacteria. Denaturing gradient gel electrophoresis (DGGE) analysis was performed to investigate the effect of both DNA isolation protocols on the lysis efficiency of bacteria in fecal samples. A higher diversity in fecal samples was observed with the bead beating method than with the Triton-X100 method. Bands in the bead beating method-derived DGGE profiles corresponding to bands of cloned sequences of the Clostridium coccoides-Eubacterium rectale group and uncultured Fusobacterium prausnitzii were absent or had low intensity in the Triton X-100 method-derived profiles. The applicability of the bead beating method was further investigated by analyzing biopsy samples from the human colon which contain approximately 10(6) cells.

Biopsy↗
PubMed2001

Cold shock proteins of Lactococcus lactis MG1363 are involved in cryoprotection and in the production of cold-induced proteins.

Members of the group of 7-kDa cold-shock proteins (CSPs) are the proteins with the highest level of induction upon cold shock in the lactic acid bacterium Lactococcus lactis MG1363. By using double-crossover recombination, two L. lactis strains were generated in which genes encoding CSPs are disrupted: L. lactis NZ9000 Delta AB lacks the tandemly orientated cspA and cspB genes, and NZ9000 Delta ABE lacks cspA, cspB, and cspE. Both strains showed no differences in growth at normal and at low temperatures compared to that of the wild-type strain, L. lactis NZ9000. Two-dimensional gel electrophoresis showed that upon disruption of the cspAB genes, the production of remaining CspE at low temperature increased, and upon disruption of cspA, cspB, and cspE, the production of CspD at normal growth temperatures increased. Northern blot analysis showed that control is most likely at the transcriptional level. Furthermore, it was established by a proteomics approach that some (non-7-kDa) cold-induced proteins (CIPs) are not cold induced in the csp-lacking strains, among others the histon-like protein HslA and the signal transduction protein LlrC. This supports earlier observations (J. A. Wouters, M. Mailhes, F. M. Rombouts, W. M. De Vos, O. P. Kuipers, and T. Abee, Appl. Environ. Microbiol. 66:3756-3763, 2000). that the CSPs of L. lactis might be directly involved in the production of some CIPs upon low-temperature exposure. Remarkably, the adaptive response to freezing by prior exposure to 10 degrees C was significantly reduced in strain NZ9000 Delta ABE but not in strain NZ9000 Delta AB compared to results with wild-type strain NZ9000, indicating a notable involvement of CspE in cryoprotection.

Adaptation, Physiological↗
PubMed2001

Flow cytometric assessment of viability of lactic acid bacteria.

The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. We investigated the usefulness of flow cytometry (FCM) for viability assessment of lactic acid bacteria. The esterase substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA binding probes propidium iodide (PI) and TOTO-1 were tested for live/dead discrimination using a Lactococcus, a Streptococcus, three Lactobacillus, two Leuconostoc, an Enterococcus, and a Pediococcus species. Plate count experiments were performed to validate the results of the FCM assays. The results showed that cFDA was an accurate stain for live cells; in exponential-phase cultures almost all cells were labeled, while 70 degrees C heat-killed cultures were left unstained. PI did not give clear live/dead discrimination for some of the species. TOTO-1, on the other hand, gave clear discrimination between live and dead cells. The combination of cFDA and TOTO-1 gave the best results. Well-separated subpopulations of live and dead cells could be detected with FCM. Cell sorting of the subpopulations and subsequent plating on agar medium provided direct evidence that cFDA labels the culturable subpopulation and that TOTO-1 labels the nonculturable subpopulation. Applied to cultures exposed to deconjugated bile salts or to acid, cFDA and TOTO-1 proved to be accurate indicators of culturability. Our experiments with lactic acid bacteria demonstrated that the combination of cFDA and TOTO-1 makes an excellent live/dead assay with versatile applications.

Bile Acids and Salts↗
PubMed2001

Analysis of the role of OpuC, an osmolyte transport system, in salt tolerance and virulence potential of Listeria monocytogenes.

The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the external environment prior to ingestion and subsequently within the animal host. Growth at high salt concentrations and low temperatures is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We utilized a novel system for generating chromosomal mutations (based on a lactococcal pWVO1-derived Ori(+) RepA(-) vector, pORI19) to identify a listerial OpuC homologue. Mutating the operon in two strains of L. monocytogenes revealed significant strain variation in the observed activity of OpuC. Radiolabeled osmolyte uptake studies, together with growth experiments in defined media, linked OpuC to carnitine and glycine betaine uptake in Listeria. We also investigated the role of OpuC in contributing to the growth and survival of Listeria in an animal (murine) model of infection. Altering OpuC resulted in a significant reduction in the ability of Listeria to colonize the upper small intestine and cause subsequent systemic infection following peroral inoculation.

ATP-Binding Cassette Transporters↗
PubMed2001

Fluorescent method for monitoring cheese starter permeabilization and lysis.

A fluorescence method to monitor lysis of cheese starter bacteria using dual staining with the LIVE/DEAD BacLight bacterial viability kit is described. This kit combines membrane-permeant green fluorescent nucleic acid dye SYTO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iodide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of Lactococcus lactis MG1363 were incubated under different conditions and subsequently labeled with SYTO 9 and PI and analyzed by flow cytometry and epifluorescence microscopy. Lysis was induced by treatment with cell wall-hydrolyzing enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes the cells. Under nonstabilizing conditions a high concentration of mutanolysin caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydrogenase. In the stabilizing buffer, mutanolysin caused membrane damage as well but the cells disintegrated at a much lower rate. Stabilizing buffer supported permeabilized cells, as indicated by a high number of PI-labeled cells. In addition, permeable cells did not release intracellular aminopeptidase N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-p-nitroanilide. Finally, with these stains and confocal scanning laser microscopy the permeabilization of starter cells in cheese could be analyzed.

Cell Membrane Permeability↗
PubMed2001

Effect of benzalkonium chloride on viability and energy metabolism in exponential- and stationary-growth-phase cells of Listeria monocytogenes.

The difference in killing exponential- and stationary-phase cells of Listeria monocytogenes by benzalkonium chloride (BAC) was investigated by plate counting and linked to relevant bioenergetic parameters. At a low concentration of BAC (8 mg liter(-1)), a similar reduction in viable cell numbers was observed for stationary-phase cells and exponential-phase cells (an approximately 0.22-log unit reduction), although their membrane potential and pH gradient were dissipated. However, at higher concentrations of BAC, exponential-phase cells were more susceptible than stationary-phase cells. At 25 mg liter(-1), the difference in survival on plates was more than 3 log units. For both types of cells, killing, i.e., more than 1-log unit reduction in survival on plates, coincided with complete inhibition of acidification and respiration and total depletion of ATP pools. Killing efficiency was not influenced by the presence of glucose, brain heart infusion medium, or oxygen. Our results suggest that growth phase is one of the major factors that determine the susceptibility of L. monocytogenes to BAC.

Anti-Infective Agents, Local↗
PubMed2000

Characterization of histatin 5 with respect to amphipathicity, hydrophobicity, and effects on cell and mitochondrial membrane integrity excludes a candidacidal mechanism of pore formation.

Histatin 5 is a 24-residue peptide from human saliva with antifungal properties. We recently demonstrated that histatin 5 translocates across the yeast membrane and targets to the mitochondria, suggesting an unusual antifungal mechanism (Helmerhorst, E. J., Breeuwer, P., van't Hof, W., Walgreen-Weterings, E., Oomen, L. C. J. M., Veerman, E. C. I., Nieuw Amerongen, A. V., and Abee, T. (1999) J. Biol. Chem. 274, 7286-7291). The present study used specifically designed synthetic analogs of histatin 5 to elucidate the role of peptide amphipathicity, hydrophobicity, and the propensity to adopt alpha-helical structures in relation to membrane permeabilization and fungicidal activity. Studies included circular dichroism measurements, evaluation of the effects on the cytoplasmic transmembrane potential and on the respiration of isolated mitochondria, and analysis of the peptide hydrophobicity/amphipathicity relationship (Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595-623). The 14-residue synthetic peptides used were dh-5, comprising the functional domain of histatin 5, and dhvar1 and dhvar4, both designed to maximize amphipathic characteristics. The results obtained show that the amphipathic analogs exhibited a high fungicidal activity, a high propensity to form an alpha-helix, dissipated the cytoplasmic transmembrane potential, and uncoupled the respiration of isolated mitochondria, similar to the pore-forming peptide PGLa (Peptide with N-terminal Glycine and C-terminal Leucine-amide). In contrast, histatin 5 and dh-5 showed fewer or none of these features. The difference in these functional characteristics between histatin 5 and dh-5 on the one hand and dhvar1, dhvar4, and PGLa on the other hand correlated well with their predicted affinity for membranes based on hydrophobicity/amphipathicity analysis. These data indicate that the salivary protein histatin 5 exerts its antifungal function through a mechanism other than pore formation.

Amino Acid Sequence↗
PubMed2000

Influence of yeast immobilization on fermentation and aldehyde reduction during the production of alcohol-free beer.

Production of alcohol-free beer by limited fermentation is optimally performed in a packed-bed reactor. This highly controllable system combines short contact times between yeast and wort with the reduction of off-flavors to concentrations below threshold values. In the present study, the influence of immobilization of yeast to DEAE-cellulose on sugar fermentation and aldehyde reduction was monitored. Immobilized cells showed higher activities of hexokinase and pyruvate decarboxylase compared to cells grown in batch culture. In addition, a higher glucose flux was observed, with enhanced excretion of main fermentation products, indicating a reduction in the flux of sugar used for biomass production. ADH activity was higher in immobilized cells compared to that in suspended cells. However, during prolonged production a decrease was observed in NAD-specific ADH activity, whereas NADP-specific activity increased in the immobilized cells. The shifts in enzyme activities and glucose flux correlate with a higher in vivo reduction capacity of the immobilized cells.

Journal Article↗
PubMed2000

Assessment of viability of microorganisms employing fluorescence techniques.

Viability assessment of microorganisms is relevant for a wide variety of applications in industry, including evaluation of inactivation treatments and quality assessment of starter cultures for beer, wine, and yoghurt production. Usually, the ability of microbial cells to reproduce is considered as the benchmark method for determination of cell viability, and this is most commonly determined by the plate count method. The time needed to form visible colonies, however, is relatively long. Therefore, there is an increasing interest in rapid methods which exploit criteria other than reproduction. In this review the applications of fluorescent probes for, e.g., determination of membrane integrity, enzyme activities, respiration, membrane potential and intracellular pH, are discussed in detail.

Bacteria↗
PubMed2000

Fluorescence assessment of Lactococcus lactis viability.

The reproduction and activity of lactic acid bacteria (LAB) are essential in their applications in the dairy industry and other fermentations. Traditionally used methods like plate counting and acidification tests require long incubation times and provide limited information. Fluorescence techniques provide possibilities for rapid assessment of cell physiology. We used traditional and fluorescence assays to assess the physiological condition of L. lactis subsp. lactis ML3 cultures that were exposed to various stress conditions. After exposure to some of the stress conditions, carboxyfluorescein (cF) labelling did not agree with plate counts. Therefore, a two-step method was developed in which cF labelling was followed by a lactose-energized efflux assay. The combined assay proved to be a good and rapid indicator for reproduction and acidification capacity of stressed L. lactis. This novel assay has potential for physiological research and dairy applications related to LAB.

Fluoresceins↗
PubMed2000

Assessment of the intracellular pH of immobilized and continuously perfused yeast cells employing fluorescence ratio imaging analysis.

The intracellular pH (pHin) of Saccharomyces cerevisiae was measured employing fluorescence ratio imaging microscopy (FRIM). The yeast cells were fluorescently labeled with the pH dependent probe 5(and-6)-carboxyfluorescein (cF) or 5(and-6)-carboxyfluorescein succinimidyl ester (cFSE), and subsequently attached to ferric nitrate pretreated glass slides. The labeled and adhered cells could still divide and were metabolically active. Measurement of the pHin was performed during continuous perfusion of the cells with buffer or medium. Cells labeled with cF are highly fluorescent and in non-energized cells the pHin could be easily measured. However, in energized yeast cells cF was accumulated in the vacuoles and/or exported to the extracellular environment, most likely by an energy-dependent transport system, thus limiting the time period over which the pHin can be effectively measured. Therefore, cFSE (which conjugates with aliphatic amines in the cytoplasm) was applied to prevent translocation of fluorescent probe to the vacuole and/or extracellular environment. The continuous perfusion in combination with the cFSE labeling of the immobilized cells was successfully applied to determine the effect of low and high pHin and addition of glucose on the pHin of individual yeast cells over a long time period.

Cells, Immobilized↗
PubMed2000

The role of cold-shock proteins in low-temperature adaptation of food-related bacteria.

There is a considerable interest in the cold adaptation of food-related bacteria, including starter cultures for industrial food fermentations, food spoilage bacteria and food-borne pathogens. Mechanisms that permit low-temperature growth involve cellular modifications for maintaining membrane fluidity, the uptake or synthesis of compatible solutes, the maintenance of the structural integrity of macromolecules and macromolecule assemblies, such as ribosomes and other components that affect gene expression. A specific cold response that is shared by nearly all food-related bacteria is the induction of the synthesis so-called cold-shock proteins (CSPs), which are small (7 kDa) proteins that are involved in mRNA folding, protein synthesis and/or freeze protection. In addition, CSPs are able to bind RNA and it is believed that these proteins act as RNA chaperones, thereby reducing the increased secondary folding of RNA at low temperatures. In this review established and novel aspects concerning the structure, function and control of these CSPs are discussed. A model for bacterial cold adaptation, with a central role for ribosomal functioning, and possible mechanisms for low-temperature sensing are discussed.

Adaptation, Biological↗