Biomedical subjects

Tjakko Abee

Publications and source records attributed to Tjakko Abee.

43 records · Page 3Linked to original sources

PubMed2002

Flow cytometric assessment of membrane integrity of ethanol-stressed Oenococcus oeni cells.

The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into the mechanisms involved in ethanol toxicity and tolerance in this organism. Exposure to ethanol resulted in an increase in the permeability of the cytoplasmic membrane, enhancing passive proton influx and concomitant loss of intracellular material (absorbing at 260 nm). Cells grown in the presence of 8% (vol/vol) ethanol revealed adaptation to ethanol stress, since these cells showed higher retention of compounds absorbing at 260 nm. Moreover, for concentrations higher than 10% (vol/vol), lower rates of passive proton influx were observed in these ethanol-adapted cells, especially at pH 3.5. The effect of ethanol on O. oeni cells was studied as the ability to efficiently retain carboxyfluorescein (cF) as an indicator of membrane integrity and enzyme activity and the uptake of propidium iodide (PI) to assess membrane damage. Flow cytometric analysis of both ethanol-adapted and nonadapted cells with a mixture of the two fluorescent dyes, cF and PI, revealed three main subpopulations of cells: cF-stained intact cells; cF- and PI-stained permeable cells, and PI-stained damaged cells. The subpopulation of O. oeni cells that maintained their membrane integrity, i.e., cells stained only with cF, was three times larger in the population grown in the presence of ethanol, reflecting the protective effect of ethanol adaptation. This information is of major importance in studies of microbial fermentations in order to assign bulk activities measured by classical methods to the very active cells that are effectively responsible for the observations.

Cell Membrane↗

PubMed2002

Enhanced levels of cold shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure.

Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations. Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP). Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4. In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L. monocytogenes LO28. After a cold shock in which the temperature was decreased from 37 degrees C to 10 degrees C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated. Pressurization of L. monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively. Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37 degrees C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques.

Bacterial Proteins↗

PubMed2002

Development of a flow cytometric method to analyze subpopulations of bacteria in probiotic products and dairy starters.

Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) and TOTO-1 [1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3dihydro(benzo-1,3-oxazole)-2-methylidene]-1-(3'-trimethylammoniumpropyl)-pyridinium tetraiodide] for (stressed) lactic acid bacteria (C. J. Bunthof, K. Bloemen, P. Breeuwer, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 67:2326-2335, 2001). cFDA stains intact cells with enzymatic activity, and TOTO-1 stains membrane-permeabilized cells. Here we used this assay to study the viability of bacterial suspensions in milk, dairy fermentation starters, and probiotic products. To facilitate FCM analysis of bacteria in milk, a commercially available milk-clearing solution was used. The procedure was optimized to increase the signal-to-noise ratio. FCM enumerations were accurate down to a concentration of 10(5) cells ml(-1). The level of retrieval of Lactobacillus plantarum WCFS 1 suspended in milk was high, and viability was not affected by the procedure. The plate counts for cleared samples of untreated cell suspensions were nearly as high as the total FCM counts, and the correlation was strong (r > 0.99). In dairy fermentation starters and in probiotic products the FCM total cell counts were substantially higher than the numbers of CFU. Three functional populations could be distinguished: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed in the products tested. This FCM method provides tools to assess the functionality of different populations in fermentation starters and probiotic products.

Animals↗

PubMed2002

Identification of proteins involved in the heat stress response of Bacillus cereus ATCC 14579.

To monitor the ability of the food-borne opportunistic pathogen Bacillus cereus to survive during minimal processing of food products, we determined its heat-adaptive response. During pre-exposure to 42 degrees C, B. cereus ATCC 14579 adapts to heat exposure at the lethal temperature of 50 degrees C (maximum protection occurs after 15 min to 1 h of pre-exposure to 42 degrees C). For this heat-adaptive response, de novo protein synthesis is required. By using two-dimensional gel electrophoresis, we observed 31 heat-induced proteins, and we determined the N-terminal sequences of a subset of these proteins. This revealed induction of stress proteins (CspB, CspE, and SodA), proteins involved in sporulation (SpoVG and AldA), metabolic enzymes (FolD and Dra), identified heat-induced proteins in related organisms (DnaK, GroEL, ClpP, RsbV, HSP16.4, YflT, PpiB, and TrxA), and other proteins (MreB, YloH, and YbbT). The upregulation of several stress proteins was confirmed by using antibodies specific for well-characterized heat shock proteins (HSPs) of B. subtilis. These observations indicate that heat adaptation of B. cereus involves proteins that function in a variety of cellular processes. Notably, a 30-min pre-exposure to 4% ethanol, pH 5, or 2.5% NaCl also results in increased thermotolerance. Also, for these adaptation processes, protein synthesis is required, and indeed, some HSPs are induced under these conditions. Collectively, these data show that during mild processing, cross-protection from heating occurs in pathogenic B. cereus, which may result in increased survival in foods.

Bacillus cereus↗

PubMed2002

Development of a standard test to assess the resistance of Staphylococcus aureus biofilm cells to disinfectants.

A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Our results show that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests.

Anti-Infective Agents, Local↗

PubMed1999

Analysis of the role of 7 kDa cold-shock proteins of Lactococcus lactis MG1363 in cryoprotection.

Low-temperature adaptation and cryoprotection were studied in the lactic acid bacterium Lactococcus lactis MG1363. An approximately 100-fold increased survival after freezing was observed when cells were shocked to 10 degrees C for 4 h compared to mid-exponential-phase cells grown at 30 degrees C, indicating an active protection against freezing. Using two-dimensional gel electrophoresis a group of 7 kDa cold-induced proteins (CSPs) was identified that corresponds to a previously described family of csp genes of L. lactis MG1363 (Wouters et al., 1998, Microbiology 144, 2885-2893). The 7 kDa CSPs appeared to be the most strongly induced proteins upon cold shock to 10 degrees C. Northern blotting and two-dimensional gel electrophoresis showed that the csp genes were maximally expressed at 10 degrees C, while induction was lower at 20 and 4 degrees C. However, pre-incubation at 20 and 4 degrees C, as well as stationary-phase conditions, also induced cryoprotection (approx. 30-, 130- and 20-fold, respectively, compared to 30 degrees C mid-exponential phase). For all treatments leading to an increased freeze survival (exposure to 4, 10 and 20 degrees C and stationary-phase conditions), increased levels of three proteins (26, 43 and 45 kDa) were observed for which a role in cryoprotection might be suggested. Increased freeze survival coincides with increased CSP expression, except for stationary-phase conditions. However, the level of observed freeze protection does not directly correlate with the csp gene expression levels. In addition, for the first time specific overproduction of a CSP in relation to freeze survival was studied. This revealed that L. lactis cells overproducing CspD at 30 degrees C show a 2-10-fold increased survival after freezing compared to control cells. This indicates that the 7 kDa cold-shock protein CspD may enhance the survival capacity after freezing but that other factors supply additional cryoprotection.

Adaptation, Physiological↗

PubMed1998

Clustered organization and transcriptional analysis of a family of five csp genes of Lactococcus lactis MG1363.

A family of genes encoding cold-shock proteins, named cspA, cspB, cspC, cspD and cspE, was cloned and sequenced from Lactococcus lactis MG1363. The genes cspA and cspB and the genes cspC and cspD are located in tandem repeats, an organization of csp genes that has never been encountered before. The five genes encode small (7.1-7.6 kDa) proteins with high mutual sequence identities (up to 85%) and high identities (about 45-65%) with the major cold-shock proteins from Escherichia coli (CspA) and Bacillus subtilis (CspB). Northern-blot analysis revealed single transcripts of about 300 nucleotides for each csp gene and showed that cspA, cspB, cspC and cspD mRNA levels were strongly increased upon cold shock to 10 degrees C (about 10-, 40-, 10- and 30-fold compared to 30 degrees C, respectively), whereas the cspE mRNA level was not increased. The expression of the cold-induced csp genes was highest in the 6-8 h lag phase after cold shock. A differential expression in time, in which cspA and cspC were maximally expressed at 2 h and cspB and cspD at 4 h after cold shock, was observed. The -35 and -10 regions of the five promoters were identified and transcriptional start sites were mapped in each case by primer extension at different temperatures which confirmed that regulation takes place at the transcriptional level. Significant differences were observed between the 5'-untranslated leader regions of the four cold-induced csp genes and the corresponding region of the non-cold-induced cspE gene.

5' Untranslated Regions↗