100,904 research outputs found
Development of a flow cytometric method to analyze subpopulations of bacteria in probiotic products and dairy starters
No full textFlow 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 105 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
Flow cytometry, fluorescent probes, and flashing bacteria
No full text Key words: fluorescent probes, flow cytometry, CSLM, viability, survival, microbial physiology, lactic acid bacteria, Lactococcus lactis , Lactobacillus plantarum , cheese, milk, probiotic In food industry there is a perceived need for rapid methods for detection and viability assessment of microbes. Fluorescent staining and flow cytometry provide excellent tools for microbial analysis. This thesis describes fluorescent techniques for assessment of the physiological state of lactic acid bacteria.Lysis of lactic acid bacteria plays a crucial role in cheese manufacturing. It is generally considered that lysis results in leakage of intracellular enzymes in the cheese curd and, thus, plays an important role in ripening and flavor formation. Bac Light (Molecular Probes) was applied for monitoring the lysis process of Lactococcus lactis MG1363 in a buffered suspension with high osmolarity to mimic cheese conditions. The Bac Light kit combines the nucleic acid dyes propidium iodide (PI) and SYTO 9. PI is commonly used to determine membrane integrity based on dye exclusion. When used in combination with the permeant SYTO 9, membrane-damaged cells are stained by PI (red) while the intact cells are stained by SYTO 9 (green). Lysis was induced with mutanolysin and followed in time using fluorescence microscopy and flow cytometry. Also, enzyme assays and plate counts were performed. The results demonstrated a transient permeable cell status that has a significant role in the lysis process. Furthermore, permeable cells were demonstrated in ripening cheese with confocal scanning laser microcopy and Bac Light.Viability assessment by conventional plate counting requires long incubation times and provides limited information. Flow cytometric assessment of the viability of lactic acid bacteria was investigated and compared with plate counts. The esterase substrate carboxyfluorescein diacetate (cFDA) and the impermeant nucleic acid dyes PI and TOTO-1 were tested using exponential phase at 70°C heat-killed cultures of a Lactococcus , a Streptococcus , three Lactobacillus , two Leuconostoc , an Enterococcus , and a Pediococcus species. The combination of cFDA and TOTO-1 gave the best results. The intact and membrane-damaged subpopulations were distinguished well. Sorting and plating showed that cFDA stained the culturable and TOTO-1 the nonculturable cells. The assay was applied to cultures exposed to deconjugated bile salts or to hydrochloric acid and results corresponded well with plate counts.Subsequently, flow cytometry with cFDA and TOTO-1 staining was applied to Lactobacillus plantarum WCFS 1 suspended in milk. To facilitate flow cytometry clearing of the milk was required. A procedure based on a milk clearing solution was optimized to increase the signal-to-noise-ratio and flow cytometry enumerations were accurate to a lower limit of 10 5cells/ml.Finally, the novel assay was applied to starter cultures for cheese and yogurt and to the probiotic products Yakult, Mona Vifit, and Orthiflorplus. Flow cytometry in combination with plate counts revealed three populations: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed between the tested products.In conclusion, the development of flow cytometry for bacteria is an important asset for microbiological research. The rapid novel methods described in this thesis provide possibilities for examination of fermentation processes and food products
Multiple deletions of the osmolyte transporters BetL, Gbu, and OpuC of Listeria monocytogenes affect virulence and growth at high osmolarity
No full textThe success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the food environment and, after ingestion, within the animal host. Growth at high salt concentrations is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We characterized L. monocytogenes LO28 strains with single, double, and triple deletions in the osmolyte transport systems BetL, Gbu, and OpuC. When single deletion mutants were tested, Gbu was found to have the most drastic effect on the rate of growth in brain heart infusion (BHI) broth with 6dded NaCl. The highest reduction in growth rate was found for the triple mutant LO28BCG (ΔbetL ΔopuC Δgbu), although the mutant was still capable of growth under these adverse conditions. In addition, we analyzed the growth and survival of this triple mutant in an animal (murine) model. LO28BCG showed a significant reduction in its ability to cause systemic infection following peroral coinoculation with the wild-type parent. Altering OpuC alone resulted in similar effects (R. D. Sleator, J. Wouters, C. G. M. Gahan, T. Abee, and C. Hill, Appl. Environ. Microbiol. 67:2692-2698, 2001), leading to the assumption that OpuC may play an important role in listerial pathogenesis. Analysis of the accumulation of osmolytes revealed that betaine is accumulated up to 300 μmol/g (dry weight) when grown in BHI broth plus 6␗aCl whereas no carnitine accumulation could be detected. Radiolabeled-betaine uptake studies revealed an inability of BGSOE (ΔbetL Δgbu) and LO28BCG to transport betaine. Indeed, for LO28BCG, no accumulated betaine was found, but carnitine was accumulated in this strain up to 600 μmol/g (dry weight) of cells, indicating the presence of a possible fourth osmolyte transporter
Impact of sorbic acid on germinant receptor-dependent and -independent germination pathways in Bacillus cereus
No full textAmino acid- and inosine-induced germination of Bacillus cereus ATCC 14579 spores was reversibly inhibited in the presence of 3 mM undissociated sorbic acid. Exposure to high hydrostatic pressure, Ca-dipicolinic acid (DPA), and bryostatin, an activator of PrkC kinase, negated this inhibition, pointing to specific blockage of signal transduction in germinant receptor-mediated germination
Letter, [Author unclear] to Paulina T. Merritt
No full textHandwritten letter to Paulina Merritt from an unknown author, October 1, 1876.
Bacillus cereus acid stress responses
No full textBacillus cereus is a ubiquitous Gram-positive organism, which frequently causes foodborne illnesses. The widespread prevalence of B. cereus makes it a common contaminant in fresh foods where it also can cause spoilage. To prevent food-borne diseases and food spoilage, foods are often processed and/or preserved. In recent years, consumers’ preferences have directed to fresher and tastier foods and this has acted as a driver for food industry to use milder processing and preservation techniques. Examples of hurdles that can be applied to preserve foods are low pH and the addition of organic acids. B. cereus may overcome these adverse conditions by displaying an adaptive stress response. The response of B. cereus upon exposure to these hurdles was investigated using two model strains, ATCC 14579 and ATCC 10987. Comparative analysis revealed numerous strain-specific genes and differences in metabolic capacities, including a urease encoding gene cluster in ATCC 10987 and a nitrate respiration cluster in ATCC 14579. A survey including ATCC 10987 and 48 environmental and outbreak-associated isolates revealed urease activity, i.e., the conversion of urea in ammonia and carbon dioxide, to be present in 10 isolates. However, the activity appeared to be too low to contribute to acid resistance in the strains tested. To search for other acid resistance mechanisms, comparative phenotype and transcriptome analyses of strains ATCC 14579 and ATCC 10987 cells exposed to organic and/or inorganic acid shocks were performed. Upon exposure to low pH with or without the addition of lactic acid or acetic acid, common acid resistance mechanisms and induction of the nitrate reductase cluster in the more acid resistant strain ATCC 14579 were revealed. Furthermore, a major oxidative response was displayed, which included the induction of several oxidative stress related genes and the production of inactivation-associated reactive oxygen species (ROS), such as hydroxyl radicals, peroxynitrite, and superoxide. ROS were detected using fluorescent probes in combination with flow cytometry, including a newly developed method using a specific probe that enables superoxide detection in Grampositive and Gram-negative bacteria. The formation of ROS was also shown upon exposure to heat and was found to be oxygen dependant. Correspondingly, assessment of B. cereus stress survival capacity revealed increased heat- and acid-resistance with cells grown and exposed to stresses in the absence of oxygen. The excess ROS may originate from stressinduced dysfunction of the aerobic electron transfer chain, which was indicated by the induction of alternative electron transfer chain components upon exposure to organic and inorganic acid shocks. Upon exposure to stress, superoxide is generated through the premature leakage of electrons to oxygen at sites in the electron transfer chain at elevated rates. Subsequently, superoxide may promote the formation of other ROS, which can cause cellular damage leading to cell death. The induction of oxidative stress related genes has been reported in numerous other studies involving a wide range of bacteria exposed to different adverse conditions. However, a clear relation between the formation of ROS and the applied environmental stress was up to now not established. Secondary oxidative responses, including the formation of ROS, are possibly common bacterial responses to severe stresses under aerobic conditions. This thesis describes genomic differences between B. cereus strains and the acid stress response of these strains on transcriptome and phenotype levels, including measurements of intracellular ROS. The findings in this study can contribute to further understanding of bacterial stress responses and secondary oxidative responses. Furthermore, the results obtained may aid to optimize and select (combinations of) stresses to apply in hurdle technology, thus enabling design of safe, milder food processing and preservation techniques. <br/
Role of ureolytic activity in Bacillus cereus nitrogen metabolism and acid survival
No full textThe presence and activities of urease genes were investigated in 49 clinical, food, and environmental Bacillus cereus isolates. Ten strains were shown to have urease genes, with eight of these strains showing growth on urea as the sole nitrogen source. Two of the urease-positive strains, including the sequenced strain ATCC 10987, could not use urea for growth, despite their capacities to produce active urease. These observations can be explained by the inability of the two strains to use ammonium as a nitrogen source. The impact of urea hydrolysis on acid stress resistance was subsequently assessed among the ureolytic B. cereus strains. However, none of the strains displayed increased fitness under acidic conditions or showed enhanced acid shock survival in the presence of urea. Expression analysis of urease genes in B. cereus ATCC 10987 revealed a low level of expression of these genes and a lack of pH-, nitrogen-, urea-, oxygen-, and growth phase-dependent modulation of mRNA transcription. This is in agreement with the low urease activity observed in strain ATCC 10987 and the other nine strains tested. Although a role for B. cereus ureolytic activity in acid survival cannot be excluded, its main role appears to be in nitrogen metabolism, where ammonium may be provided to the cells in nitrogen-limited, urea-containing environment
Comparative transcriptomic and phenotypic analysis of the responses of Bacillus cereus to various disinfectant treatments
No full textAntimicrobial chemicals are widely applied to clean and disinfect food-contacting surfaces. However, the cellular response of bacteria to various disinfectants is unclear. In this study, the physiological and genome-wide transcriptional responses of Bacillus cereus ATCC 14579 exposed to four different disinfectants (benzalkonium chloride, sodium hypochlorite, hydrogen peroxide, and peracetic acid) were analyzed. For each disinfectant, concentrations leading to the attenuation of growth, growth arrest, and cell death were determined. The transcriptome analysis revealed that B. cereus, upon exposure to the selected concentrations of disinfectants, induced common and specific responses. Notably, the common response included genes involved in the general and oxidative stress responses. Exposure to benzalkonium chloride, a disinfectant known to induce membrane damage, specifically induced genes involved in fatty acid metabolism. Membrane damage induced by benzalkonium chloride was confirmed by fluorescence microscopy, and fatty acid analysis revealed modulation of the fatty acid composition of the cell membrane. Exposure to sodium hypochlorite induced genes involved in metabolism of sulfur and sulfur-containing amino acids, which correlated with the excessive oxidation of sulfhydryl groups observed in sodium hypochlorite-stressed cells. Exposures to hydrogen peroxide and peracetic acid induced highly similar responses, including the upregulation of genes involved in DNA damage repair and SOS response. Notably, hydrogen peroxide- and peracetic acid-treated cells exhibited high mutation rates correlating with the induced SOS respons
Bacillus cereus responses to acid stress
No full textCoping with acid environments is one of the prerequisites for the soil saprophytic and human pathogenic lifestyle of Bacillus cereus. This minireview highlights novel insights in the responses displayed by vegetative cells and germinating spores of B. cereus upon exposure to low pH as well as organic acids, including acetic acid, lactic acid and sorbic acid. Insights regarding the possible acid-inflicted damage, physiological responses and protective mechanisms have been compiled based on single cell fluorescence microscopy, flow cytometry and transcriptome analyses
Two-component signal transduction in Bacillus cereus and closely related bacteria
No full textBacillus cereus is a Gram-positive pathogen that is recognised as an important cause of food-borne disease worldwide. Within the genus Bacillus, B. cereus and its closest relatives form a homogeneous subdivision that has been termed the B. cereus group. This group includes B. anthracis, a pathogen that can cause anthrax in mammals, and B. thuringiensis, an insect pathogen that is used as an insecticide worldwide. Members of the B. cereus group can adapt to a wide range of environmental challenges. In bacteria, these challenges are generally monitored by two-component systems (TCS), which consist of a histidine kinase (HK) and a partner response regulator (RR). Upon sensing a specific environmental stimulus, the HK activates its cognate RR, which in turn controls the expression of genes that are involved in the appropriate response. This thesis describes the functional analysis of TCSs in the B. cereus group. By using in silico techniques, 50-58 HKs and 48-52 RRs were identified in eight different B. cereus group genomes. Biological functions, including the involvement in sporulation, biofilm formation and host-microbe interactions were predicted for these TCS proteins. A phylogenetic footprinting approach was developed and used to identify specific binding sites and target genes for over 50% of the B. cereus group DNA-binding RRs. These predictions allowed relating several RRs to a minimal regulon and thereby to a characteristic transcriptional response. To further support these predictions, the transcriptomes of two B. cereus TCS deletion mutants (ΔyvrHG and ΔyufLM) were analysed and compared with the transcriptome of wild-type B. cereus. This revealed that the minimal regulon predictions were correct for the two respective TCSs. Furthermore, the predicted biological roles for these TCSs, including roles in antibiotic resistance (YvrHG) and fumarate metabolism (YufLM), were supported by phenotypic tests. Besides the many “classical” HKs and RRs detected in the B. cereus group, several a-typical TCS proteins were found. These included five RRs without a DNA-binding output domain and two hybrid HKs (HK-RR fusions). Genome analyses revealed that one of the hybrid HK-encoding genes (BC1008) is located in a conserved gene cluster that also encodes the a-typical RR RsbY. In B. cereus, RsbY is known to activate the key stress-responsive sigma factor σB. As a partner HK for RsbY was still “missing”, the role of BC1008 in the σB-mediated stress response was tested. Indeed, a bc1008 deletion strain appeared incapable of inducing σB and its associated regulon upon stress conditions and appeared impaired in its heat adaptive response. In addition, truncation of the BC1008 fused RR receiver domain indicated that this domain plays a role in fine-tuning BC1008 activity. A comparative genome analysis further indicated that BC1008-type hybrid HKs control σB-like sigma factors in at least several other Gram-positive bacteria, including Geobacillus, Paenibacillus and actinobacteria. In summary, the research described in this thesis contributes to our understanding of B. cereus adaptive responses through TCSs. This knowledge may be applied for the development of novel intervention strategies for an improved control of B. cereus in food production environments. <br/
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