Biomedical subjects

Henrik Siegumfeldt

Publications and source records attributed to Henrik Siegumfeldt.

11 recordsLinked to original sources

PubMed2006

Listeria innocua and Lactobacillus delbrueckii subsp. bulgaricus employ different strategies to cope with acid stress.

Responses of Listeria innocua and Lactobacillus delbrueckii subsp. bulgaricus to a rapid change in extracellular pH (pHex) from pHex 6 to a range of concentrations down to pHex 3.0 were examined, using HCl and lactic acid (LA) as acidulants. A new fluorescent probe 5-(and-6)-carboxy-2', 7'-dichlorofluorescein diacetate succinimidyl ester (CDCFDA-SE) was employed that enabled reliable measurements of intracellular pH (pHi) to a minimum pHi of 4.0. Changes in pHi and H+ fluxes from immobilised bacteria were measured using fluorescence ratio imaging microscopy (FRIM) and a non-invasive ion flux measuring technique (MIFE), respectively. L. innocua maintained a relatively constant pHi of 5.5-6.1 at pHex 4 and 5 via H+ extrusion. In contrast, L. delbrueckii subsp. bulgaricus progressively lowered pHi towards pHex over the entire pHex range examined. The type of acidulant used influenced pH regulation with both pHi and H+ -fluxes being more severely affected by LA compared to HCl. Overall, our data demonstrated different adaptive strategies in these two bacteria. While L. innocua expels protons to maintain a constant pHi, L. delbrueckii subsp. bulgaricus allows proton entry after acidic treatment so that pHi follows pHex.

Adaptation, Physiological↗

PubMed2006

Leucocins 4010 from Leuconostoc carnosum cause a matrix related decrease in intracellular pH of Listeria monocytogenes.

A mixed culture of single cells of Listeria monocytogenes and the bacteriocin producing Leuconostoc carnosum 4010 showed growth inhibition of L. monocytogenes, although the intracellular pH (pHi) of L. monocytogenes followed by fluorescence ratio imaging microscopy was not affected. Furthermore, L. monocytogenes was exposed to the bacteriocins leucocins 4010 and nisin either in a liquid filled chamber or on the surface of an agar containing bacteriocins. Both bacteriocins caused dissipation of the pH gradient in L. monocytogenes and the effect was clearly dependent on the matrix, as the decrease in pHi occurred much more rapidly in liquid than in agar.

Bacteriocins↗

PubMed2006

Differential expression of proteins and genes in the lag phase of Lactococcus lactis subsp. lactis grown in synthetic medium and reconstituted skim milk.

We investigated protein and gene expression in the lag phase of Lactococcus lactis subsp. lactis CNRZ 157 and compared it to the exponential and stationary phases. By means of two-dimensional polyacrylamide gel electrophoresis, 28 highly expressed lag-phase proteins, implicated in nucleotide metabolism, glycolysis, stress response, translation, transcription, cell division, amino acid metabolism, and coenzyme synthesis, were identified. Among the identified proteins, >2-fold induction and down-regulation in the lag phase were determined for 12 proteins in respect to the exponential phase and for 18 proteins in respect to the stationary phase. Transcriptional changes of the lag-phase proteins in L. lactis were studied by oligonucleotide microarrays. Good correlation between protein and gene expression studies was demonstrated for several differentially expressed proteins, including nucleotide biosynthetic enzymes, adenylosuccinate synthase (PurA), IMP dehydrogenase (GuaB), and aspartate carbamoyl transferase (PyrB); heat-shock protein DnaK; serine hydroxymethyl transferase (GlyA); carbon catabolite control protein (CcpA); elongation factor G (FusA); and cell division protein (FtsZ).

Animals↗

PubMed2006

Two subpopulations of Listeria monocytogenes occur at subinhibitory concentrations of leucocin 4010 and nisin.

In situ analyses of single Listeria monocytogenes cells at subinhibitory concentrations of leucocin 4010 and nisin revealed two subpopulations when measured by fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester. One subpopulation consisted of cells with a dissipated pH gradient (DeltapH), and the other consisted of cells that maintained DeltapH. The proportion of cells belonging to each subpopulation was estimated, and the concentrations of bacteriocins required to dissipate DeltapH for 90% of the cell population (ED90) was predicted. ED90 increased after the addition of sodium chloride (1 to 3% [wt/vol]) to the bacteriocin solutions, while ED90 decreased by the addition of sodium nitrite (60 and 100 ppm). Other meat additives, including sodium phosphate, sodium lactate, sodium citrate, and sodium acetate slightly increased ED90. The inhibitory effect of sodium chloride on the antilisterial activity of leucocin 4010 and nisin was confirmed on the surfaces of meat sausages. This study highlights the important practical implications of applying subinhibitory concentrations of bacteriocins, which results in unaffected target cells. In situ analyses by FRIM in combination with modeling of single-cell data can be applied to ensure that sufficient concentrations of bacteriocins are used in food preservation.

Bacteriocins↗

PubMed2005

Interactive optical trapping shows that confinement is a determinant of growth in a mixed yeast culture.

Applying a newly developed user-interactive optical trapping system, we controllably surrounded individual cells of one yeast species, Hanseniaspora uvarum, with viable cells of another yeast species, Saccharomyces cerevisiae, thus creating a confinement of the former. Growth of surrounded and non-surrounded H. uvarum cells was followed under a microscope by determining their generation time. The average generation time of surrounded H. uvarum cells was 15% higher than that of non-surrounded cells, thereby showing that the confinement imposed by viable S. cerevisiae cells on H. uvarum inhibits growth of the latter. This study is the first to demonstrate that confinement is a determinant of growth in a microbial ecosystem.

Culture Media↗

PubMed2004

Osmotic stress leads to decreased intracellular pH of Listeria monocytogenes as determined by fluorescence ratio-imaging microscopy.

Intracellular pH (pH(i)) of Listeria monocytogenes was determined after exposure to NaCl or sorbitol in liquid and solid media (agar). Both compounds decreased pH(i), and recovery on solid medium was impaired compared to that in liquid medium. N,N'-dicyclohexylcarbodiimide abolished pH(i) recovery, and lowering a(w) with glycerol showed no effect on pH(i).

Culture Media↗

PubMed2002

Responses of Listeria monocytogenes to acid stress and glucose availability monitored by measurements of intracellular pH and viable counts.

Physiological aspects of the response of Listeria monocytogenes to acidic conditions and effect of glucose availability were studied by fluorescence ratio-imaging microscopy (FRIM) as compared with traditional viable counts. Three types of experiments were conducted: (i) static with measurements of intracellular pH (pHi) at extracellular pH (pHo) values ranging from pH 3.0 to 6.0 at 0.5 pH unit intervals; (ii) kinetic with monitoring of bacterial responses to changes in the pHo from the value of 6.0 to 4.0 or 3.0; (iii) survival experiments studying bacterial recovery in response to a shift to favourable conditions after a treatment at low pH. All the experiments were performed at three levels of glucose in the medium (0, 1, and 10 mM). Both survival and pHi were greatly affected by pHo and glucose availability with the highest values for CFU and pHi at highest glucose concentration and pHo values in the medium in all trials. A high correlation (R2 = 0.995) between pHi and CFU counts was observed. The pH gradient started to collapse at pHo 4 and below for trials with glucose in the medium and at pHo 5.5 and below without glucose. A recovery step was proposed after the apparently lethal treatment to assess cell viability by FRIM.

Cells, Immobilized↗

PubMed2002

Rapid assessment of cell viability of Lactobacillus delbrueckii subsp. bulgaricus by measurement of intracellular pH in individual cells using fluorescence ratio imaging microscopy.

The aim of this study was to investigate if the measurement of intracellular pH (pHi) of individual cells by fluorescence ratio imaging microscopy (FRIM) could be utilized as a rapid method for determining the bacterial viability, using Lactobacillus delbrueckii subsp. bulgaricus as a model organism. Five different standardized cultures with equal cell densities but varying viability were prepared on a trial-to-trial basis by combining aliquots of frozen and lyophilized cells with a 50-fold difference in viability, determined by the ability to form colonies on solid growth media. The acidification of milk and Acidification Power Test were used to determine the activity of these cultures. As expected, the cultures containing a higher proportion of viable cells acidified milk faster and performed better in the Acidification Power Test. All cells were fluorescent after staining with carboxyfluorescein diacetate succinimidyl ester but frozen cells reached higher fluorescence intensities than lyophilized cells. The number of strongly fluorescent cells determined by flow cytometry exceeded the number of viable cells determined by CFU. Analysis of pHi of individual cells by FRIM at an extracellular pH of 6.0 revealed two populations of cells with an average pHi of 6.9 +/- 0.1 and 6.1 +/- 0.1. As the number of cells maintaining a pH-gradient of 0.9 +/- 0.1 correlated well with CFU, we suggest that FRIM can be used as a rapid method for the determination of viability of L. delbrueckii subsp. bulgaricus. Measurement of pHi on a single cell basis is expected to provide accurate prediction of the fermentation performance in a wider range of industrial fermentation.

Colony Count, Microbial↗

PubMed2002

Responses of Listeria monocytogenes to acid stress and glucose availability revealed by a novel combination of fluorescence microscopy and microelectrode ion-selective techniques.

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H(+) at either side of the bacterial membrane in real time and therefore to evaluate the rate of H(+) flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH(o)) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH(i)) and net H(+) fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH(i) remained above 7.0 within a pH(o) range of 4 to 6 and decreased below pH(o) 4. Above pH(o) 4, H(+) extrusion increased correspondingly, with the maximum value at pH(o) 5.5, and below pH(o) 4, a net H(+) influx was observed. Without glucose in the medium, the pH(i) decreased, and a net H(+) influx was observed below pH(o) 5.5. A high correlation (R = 0.75 to 0.92) between the pH(i) and net H(+) flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH(i) homeostasis in L. monocytogenes subjected to acid stress.

Cells, Immobilized↗

PubMed2002

Noninvasive measurement of bacterial intracellular pH on a single-cell level with green fluorescent protein and fluorescence ratio imaging microscopy.

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pHi) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pHi, allowing fast and noninvasive determination of pHi that is ideally suited for direct analysis of individual bacterial cells present in complex environments.

Escherichia coli↗

PubMed1999

Use of fluorescence ratio imaging for intracellular pH determination of individual bacterial cells in mixed cultures.

The development of a rapid method for measuring intracellular pH (pHi) in single bacterial cells is described. Lactobacillus delbrueckii subsp. bulgaricus and Listeria innocua were used as test organisms. The method is based upon fluorescence microscopy and ratio imaging of cells stained with carboxyfluorescein succinimidyl ester. After staining, the bacteria were immobilized on a membrane filter and transferred to a closed perfusion chamber, allowing control of the cell environment during analysis. The set-up was optimized with regard to the use of neutral-density filters and background subtraction, for determining the excitation ratio 490 nm/435 nm (R490/435) independent of the excitation light intensity, and to reduce photobleaching. This allowed for time-lapse studies with multiple exposures. To study the pHi of Lb. delbrueckii subsp. bulgaricus and L. innocua in response to different extracellular pH (pHex) values, an in vivo calibration curve was constructed in the pHi range 5.0-8.5. Distinct differences between the two cultures were observed. L. innocua maintained a near-neutral pHi almost independently of pHex (5.0-8.0), whereas the pHi of Lb. delbrueckii subsp. bulgaricus decreased with decreasing pHex. In pure and mixed cultures at pHex 5.0, the pHi values of Lb. delbrueckii subsp. bulgaricus and L. innocua were 6.1 +/- 0.2 and 7.5 +/- 0.2, respectively. This difference in pHi may explain how Lb. delbrueckii subsp. bulgaricus obtains a competitive advantage over L. innocua at low pHex.

Culture Media↗