Biomedical subjects

Tjakko Abee

Publications and source records attributed to Tjakko Abee.

At least 37 records · Page 2Linked to original sources

PubMed2005

Identification of novel Salmonella enterica serovar Typhimurium DT104-specific prophage and nonprophage chromosomal sequences among serovar Typhimurium isolates by genomic subtractive hybridization.

Genomic subtractive hybridization was performed between Salmonella enterica serovar Typhimurium LT2 and DT104 to search for novel Salmonella serovar Typhimurium DT104-specific sequences. The subtraction resulted mainly in the isolation of DNA fragments with sequence similarity to phages. Two fragments identified were associated with possible virulence factors. One fragment was identical to irsA of Salmonella serovar Typhimurium ATCC 14028, which is suggested to be involved in macrophage survival. The other fragment was homologous to HldD, an Escherichia coli O157:H7 lipopolysaccharide assembly-related protein. Five selected DNA fragments-irsA, the HldD homologue, and three fragments with sequence similarity to prophages-were tested for their presence in 17 Salmonella serovar Typhimurium DT104 isolates and 27 non-DT104 isolates by PCR. All five selected DNA fragments were Salmonella serovar Typhimurium DT104 specific among the serovar Typhimurium isolates tested. These DNA fragments can be useful for better detection and typing of Salmonella serovar Typhimurium DT104.

Bacteriophage Typing↗

PubMed2005

Analysis of the role of RsbV, RsbW, and RsbY in regulating {sigma}B activity in Bacillus cereus.

The alternative sigma factor sigma(B) is an important regulator of the stress response of Bacillus cereus. Here, the role of the regulatory proteins RsbV, RsbW, and RsbY in regulating sigma(B) activity in B. cereus is analyzed. Functional characterization of RsbV and RsbW showed that they act as an anti-sigma factor antagonist and an anti-sigma factor, respectively. RsbW can also act as a kinase on RsbV. These data are in line with earlier functional characterizations of RsbV and RsbW homologs in B. subtilis. The rsbY gene is unique to B. cereus and its closest relatives and is predicted to encode a protein with an N-terminal CheY domain and a C-terminal PP2C domain. In an rsbY deletion mutant, the sigma(B) response upon stress exposure was almost completely abolished, but the response could be restored by complementation with full-length rsbY. Expression analysis showed that rsbY is transcribed from both a sigma(A)-dependent promoter and a sigma(B)-dependent promoter. The central role of RsbY in regulating the activity of sigma(B) indicates that in B. cereus, the sigma(B) activation pathway is markedly different from that in other gram-positive bacteria.

Bacillus cereus↗

PubMed2004

Using Lactococcus lactis for glutathione overproduction.

Glutathione and gamma-glutamylcysteine were produced in Lactococcus lactis using a controlled expression system and the genes gshA and gshB from Escherichia coli encoding the enzymes gamma-glutamylcysteine synthetase and glutathione synthetase. High levels of gamma-glutamylcysteine were found in strains growing on chemically defined medium and expressing either gshA alone or both gshA and gshB. As anticipated, glutathione was found in a strain expressing gshA and gshB. The level of glutathione production could be increased by addition of the precursor amino acid cysteine to the medium. The addition of cysteine led to an increased activity of glutathione synthetase, which is remarkable because the amino acid is not a substrate of this enzyme. The final intracellular glutathione concentration attained was 358 nmol mg(-1) protein, which is the highest concentration reported for a bacterium, demonstrating the suitability of engineered L. lactis for fine-chemical production and as a model for studies of the impact of glutathione on flavour formation and other properties of food.

Cloning, Molecular↗

PubMed2004

Impact of genomics on microbial food safety.

Genome sequences are now available for many of the microbes that cause food-borne diseases. The information contained in pathogen genome sequences, together with the development of themed and whole-genome DNA microarrays and improved proteomics techniques, might provide tools for the rapid detection and identification of such organisms, for assessing their biological diversity and for understanding their ability to respond to stress. The genomic information also provides insight into the metabolic capacity and versatility of microbes; for example, specific metabolic pathways might contribute to the growth and survival of pathogens in a range of niches, such as food-processing environments and the human host. New concepts are emerging about how pathogens function, both within foods and in interactions with the host. The future should bring the first practical benefits of genome sequencing to the field of microbial food safety, including strategies and tools for the identification and control of emerging pathogens.

Biofilms↗

PubMed2004

Growth and sporulation of Bacillus cereus ATCC 14579 under defined conditions: temporal expression of genes for key sigma factors.

An airlift fermentor system allowing precise regulation of pH and aeration combined with a chemically defined medium was used to study growth and sporulation of Bacillus cereus ATCC 14579. Sporulation was complete and synchronous. Expression of sigA, sigB, sigF, and sigG was monitored with real-time reverse transcription-PCR, and the pattern qualitatively resembled that of Bacillus subtilis. This method allows reproducible production of stable spores, while the synchronous growth and defined conditions are excellently suitable for further gene expression studies of cellular differentiation of B. cereus.

Bacillus cereus↗

PubMed2004

Effect of adaptation to ethanol on cytoplasmic and membrane protein profiles of Oenococcus oeni.

The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into mechanisms of ethanol toxicity and of acquired ethanol tolerance in this organism. Therefore, the site-specific location of proteins involved in ethanol adaptation, including cytoplasmic, membrane-associated, and integral membrane proteins, was investigated. Ethanol triggers alterations in protein patterns of O. oeni cells stressed with 12% ethanol for 1 h and those of cells grown in the presence of 8% ethanol. Levels of inosine-5'-monophosphate dehydrogenase and phosphogluconate dehydrogenase, which generate reduced nicotinamide nucleotides, were decreased during growth in the presence of ethanol, while glutathione reductase, which consumes NADPH, was induced, suggesting that maintenance of the redox balance plays an important role in ethanol adaptation. Phosphoenolpyruvate:mannose phosphotransferase system (PTS) components of mannose PTS, including the phosphocarrier protein HPr and EII(Man), were lacking in ethanol-adapted cells, providing strong evidence that mannose PTS is absent in ethanol-adapted cells, and this represents a metabolic advantage to O. oeni cells during malolactic fermentation. In cells grown in the presence of ethanol, a large increase in the number of membrane-associated proteins was observed. Interestingly, two of these proteins, dTDT-glucose-4,6-dehydratase and D-alanine:D-alanine ligase, are known to be involved in cell wall biosynthesis. Using a proteomic approach, we provide evidence for an active ethanol adaptation response of O. oeni at the cytoplasmic and membrane protein levels.

Adaptation, Physiological↗

PubMed2004

Germination of penicillium paneum Conidia is regulated by 1-octen-3-ol, a volatile self-inhibitor.

Penicillium paneum is an important contaminant of cereal grains which is able to grow at low temperature, low pH, high levels of carbon dioxide, and under acid conditions. P. paneum produces mycotoxins, which may be harmful to animals and humans. We found that conidia in dense suspensions showed poor germination, suggesting the presence of a self-inhibitor. A volatile compound(s) produced by these high-density conditions also inhibited mycelial growth of different species of fungi belonging to a variety of genera, suggesting a broad action range. The heat-stable compound was isolated by successive centrifugation of the supernatant obtained from spore suspensions with a density of 10(9) conidia ml(-1). By using static headspace analyses, two major peaks were distinguished, with the highest production of these metabolites after 22 h of incubation at 25 degrees C and shaking at 140 rpm. Gas chromatography coupled with mass spectra analysis revealed the compounds to be 3-octanone and 1-octen-3-ol. Notably, only the latter compound appeared to block the germination process at different developmental stages of the conidia (swelling and germ tube formation). In this study, 1-octen-3-ol influenced different developmental processes during the P. paneum life cycle, including induction of microcycle conidiation and inhibition of spore germination. Therefore, the compound can be considered a fungal hormone during fungal development.

Culture Media↗

PubMed2004

Molecular and physiological analysis of the role of osmolyte transporters BetL, Gbu, and OpuC in growth of Listeria monocytogenes at low temperatures.

Listeria monocytogenes is a ubiquitous food-borne pathogen found widely distributed in nature as well as an undesirable contaminant in a variety of fresh and processed foods. This ubiquity can be at least partly explained by the ability of the organism to grow at high osmolarity and reduced temperatures, a consequence of its ability to accumulate osmo- and cryoprotective compounds termed osmolytes. Single and multiple deletions of the known osmolyte transporters BetL, Gbu, and OpuC significantly reduce growth at low temperatures. During growth in brain heart infusion broth at 7 degrees C, Gbu and OpuC had a more pronounced role in cryoprotection than did BetL. However, upon the addition of betaine to defined medium, the hierarchy of transporter importance shifted to Gbu > BetL > OpuC. Upon the addition of carnitine, only OpuC appeared to play a role in cryoprotection. Measurements of the accumulated osmolytes showed that betaine is preferred over carnitine, while in the absence of a functional Gbu, carnitine was accumulated to higher levels than betaine was at 7 degrees C. Transcriptional analysis of the genes encoding BetL, Gbu, and OpuC revealed that each transporter is induced to different degrees upon cold shock of L. monocytogenes LO28. Additionally, despite being transcriptionally up-regulated upon cold shock, a putative fourth osmolyte transporter, OpuB (identified by bioinformatic analysis and encoded by lmo1421 and lmo1422), showed no significant contribution to listerial chill tolerance. Growth of the quadruple mutant LO28deltaBCGB (deltabetL deltaopuC deltagbu deltaopuB) was comparable to the that of the triple mutant LO28deltaBCGsoe (deltabetL deltaopuC deltagbu) at low temperatures. Here, we conclude that betaine and carnitine transport upon low-temperature exposure is mediated via three osmolyte transporters, BetL, Gbu, and OpuC.

ATP-Binding Cassette Transporters↗

PubMed2004

Identification of sigma factor sigma B-controlled genes and their impact on acid stress, high hydrostatic pressure, and freeze survival in Listeria monocytogenes EGD-e.

The gene encoding the alternative sigma factor sigma(B) in Listeria monocytogenes is induced upon exposure of cells to several stresses. In this study, we investigated the impact of a sigB null mutation on the survival of L. monocytogenes EGD-e at low pH, during high-hydrostatic-pressure treatment, and during freezing. The survival of Delta sigB mutant exponential-phase cells at pH 2.5 was 10,000-fold lower than the survival of EGD-e wild-type cells. Moreover, the Delta sigB mutant failed to show an acid tolerance response. Upon preexposure for 1 h to pH 4.5, the survival at pH 2.5 was 100,000-fold lower for the Delta sigB mutant than for the wild type. The glutamate decarboxylase (GAD) acid resistance system is important in survival and adaptation of L. monocytogenes in acidic conditions. The sigma(B) dependence of the gad genes (gadA, gadB, gadC, gadD, and gadE) was analyzed in silico. Putative sigma(B)-dependent promoter sites were found upstream of the gadCB operon (encoding a glutamate/gamma-aminobutyrate antiporter and a glutamate decarboxylase, respectively) and the lmo2434 gene (gadD, encoding a putative glutamate decarboxylase). Reverse transcriptase PCR revealed that expression of the gadCB operon and expression of gadD are indeed sigma(B) dependent. In addition, a proteomics approach was used to analyze the protein expression profiles upon acid exposure. Although the GAD proteins were not recovered, nine proteins accumulated in the wild type but not in the Delta sigB strain. These proteins included Pfk, GalE, ClpP, and Lmo1580. Exposure to pH 4.5, in order to preload cells with active sigma(B) and consequently with sigma (B)-dependent general stress proteins, also provided considerable protection against high-hydrostatic-pressure treatment and freezing. The combined data argue that the expression of sigma(B)-dependent genes provides L. monocytogenes with nonspecific multiple-stress resistance that may be relevant for survival in the natural environment as well as during food processing.

Bacterial Proteins↗

PubMed2004

Identification of sigmaB-dependent genes in Bacillus cereus by proteome and in vitro transcription analysis.

The alternative sigma factor sigmaB of the food pathogen Bacillus cereus is activated upon stress exposure and plays a role in the adaptive response of vegetative cells. This study describes the identification of sigmaB-dependent genes in B. cereus. Two-dimensional gel electrophoresis was performed with protein extracts from a sigmaB-overproducing B. cereus strain. Nine protein spots, which were absent from the negative control, were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry or N-terminal sequencing. The sigmaB-dependent expression of the corresponding genes was confirmed by Northern blot analysis with RNA isolated from B. cereus ATCC 14579 and its sigB null mutant. Northern blot analysis also revealed that six other genes were part of sigmaB-dependent operons. The proteins that are predicted to be encoded by the sigmaB-dependent genes include an intracellular protease, a Mg2+ transporter, and a thiamine biosynthesis protein (ThiG). Highly conserved promoter sites were found to precede all sigmaB-dependent genes, with the exception of thiG. By searching the B. cereus genome for this conserved promoter sequence, five more candidate sigmaB-dependent genes were identified. Northern blot analysis and in vitro transcription experiments with a reconstituted B. cereus sigmaB-RNA polymerase holoenzyme confirmed the sigmaB dependency of two of these genes and strongly suggested that two other genes, encoding an oligopeptide-binding OppA-like protein and subunit II of the cytochrome d ubiquinol oxidase, are also sigmaB dependent. In conclusion, sigmaB of B. cereus not only regulates genes directly involved in the stress response but may also control specific metabolic rearrangements.

Bacillus cereus↗

PubMed2004

The alternative sigma factor sigmaB of Bacillus cereus: response to stress and role in heat adaptation.

A gene cluster encoding the alternative sigma factor sigma(B), three predicted regulators of sigma(B) (RsbV, RsbW, and RsbY), and one protein whose function is not known (Orf4) was identified in the genome sequence of the food pathogen Bacillus cereus ATCC 14579. Western blotting with polyclonal antibodies raised against sigma(B) revealed that there was 20.1-fold activation of sigma(B) after a heat shock from 30 to 42 degrees C. Osmotic upshock and ethanol exposure also upregulated sigma(B), albeit less than a heat shock. When the intracellular ATP concentration was decreased by exposure to carbonyl cyanide m-chlorophenylhydrazone (CCCP), only limited increases in sigma(B) levels were observed, revealing that stress due to ATP depletion is not an important factor in sigma(B) activation in B. cereus. Analysis of transcription of the sigB operon by Northern blotting and primer extension revealed the presence of a sigma(B)-dependent promoter upstream of the first open reading frame (rsbV) of the sigB operon, indicating that transcription of sigB is autoregulated. A second sigma(B)-dependent promoter was identified upstream of the last open reading frame (orf4) of the sigB operon. Production of virulence factors and the nonhemolytic enterotoxin Nhe in a sigB null mutant was the same as in the parent strain. However, sigma(B) was found to play a role in the protective heat shock response of B. cereus. The sigB null mutant was less protected against the lethal temperature of 50 degrees C by a preadaptation to 42 degrees C than the parent strain was, resulting in a more-than-100-fold-reduced survival of the mutant after 40 min at 50 degrees C.

Adaptation, Physiological↗

PubMed2003

The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence.

A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183-3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRDeltaGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRDeltaGly mutants than in the wt strain, indicative of the CtsRDeltaGly protein being inactive. Further evidence that the CtsRDeltaGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRDeltaGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRDeltaGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.

Adenosine Triphosphatases↗

PubMed2003

Glutathione protects Lactococcus lactis against oxidative stress.

Glutathione was found in several dairy Lactococcus lactis strains grown in M17 medium. None of these strains was able to synthesize glutathione. In chemically defined medium, L. lactis subsp. cremoris strain SK11 was able to accumulate up to approximately 60 mM glutathione when this compound was added to the medium. Stationary-phase cells of strain SK11 grown in chemically defined medium supplemented with glutathione showed significantly increased resistance (up to fivefold increased resistance) to treatment with H2O2 compared to the resistance of cells without intracellular glutathione. The resistance to H2O2 treatment was found to be dependent on the accumulation of glutathione in 16 strains of L. lactis tested. We propose that by taking up glutathione, L. lactis might activate a glutathione-glutathione peroxidase-glutathione reductase system in stationary-phase cells, which catalyzes the reduction of H2O2. Glutathione reductase, which reduces oxidized glutathione, was detectable in most strains of L. lactis, but the activities of different strains were very variable. In general, the glutathione reductase activities of L. lactis subsp. lactis are higher than those of L. lactis subsp. cremoris, and the activities were much higher when strains were grown aerobically. In addition, glutathione peroxidase is detectable in strain SK11, and the level was fivefold greater when the organism was grown aerobically than when the organism was grown anaerobically. Therefore, the presence of glutathione in L. lactis could result in greater stability under storage conditions and quicker growth upon inoculation, two important attributes of successful starter cultures.

Culture Media↗

PubMed2003

Membrane fluidity adjustments in ethanol-stressed Oenococcus oeni cells.

The effect of ethanol on the cytoplasmic membrane of Oenococcus oeni cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells. To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy. Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface. Fluidity increased with increasing concentrations of ethanol. Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity. Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol. Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage. This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved. We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested. We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface.

Adaptation, Physiological↗

PubMed2002

Analysis of the heat-adaptive response of psychrotrophic Bacillus weihenstephanensis.

The heat-adaptive response of the psychrotrophic spoilage bacterium Bacillus weihenstephanensis DSM11827 is described. It is demonstrated that vegetative cells of B. weihenstephanensis adapts to heat exposure at 47 degrees C by prior exposure to heat at the nonlethal temperature of 38 degrees C. For this adaptive response, protein synthesis is required and maximum adaptation was noted after 15 min to 2 h prior exposure at 38 degrees C. By using two-dimensional gel electrophoresis (2D-E), an overview of the heat-shock proteins (HSPs) of B. weihenstephanensis was obtained and it was shown that the production of 15 proteins increased upon exposure to 38 degrees C. In more detail, the use of specific antibodies revealed induction of the HSPs DnaK, DnaJ, GroEL, ClpC, ClpP and ClpX of B. weihenstephanensis. In addition, also pre-exposure to other stresses than heat, such as exposure to a high salt concentration, low pH, a high ethanol concentration or low temperature, resulted in development of increased heat tolerance of B. weihenstephanensis, and during these conditions, an increased production of some HSPs was noted. This phenomenon of cross-protection might be of substantial importance in relation to the design of safe minimal processing regimes.

Adaptation, Physiological↗

PubMed2002

Assessment of the microbody luminal pH in the filamentous fungus Penicillium chrysogenum.

The enzymes of the penicillin biosynthetic pathway in Penicillium chrysogenum are located in different subcellular compartments. Consequently, penicillin pathway precursors and the biologically active penicillins have to cross one or more membranes. The final enzymatic step that is mediated by acyltransferase takes place in a microbody. The pH of the microbody lumen in penicillin producing cells has been determined with fluorescent probes and mutants of the green fluorescent protein and found to be slightly alkaline.

Fluorescent Dyes↗

PubMed2002

Multiple deletions of the osmolyte transporters BetL, Gbu, and OpuC of Listeria monocytogenes affect virulence and growth at high osmolarity.

The 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 6% added NaCl. The highest reduction in growth rate was found for the triple mutant LO28BCG (DeltabetL DeltaopuC Deltagbu), 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 micro mol/g (dry weight) when grown in BHI broth plus 6% NaCl whereas no carnitine accumulation could be detected. Radiolabeled-betaine uptake studies revealed an inability of BGSOE (DeltabetL Deltagbu) and LO28BCG to transport betaine. Indeed, for LO28BCG, no accumulated betaine was found, but carnitine was accumulated in this strain up to 600 micro mol/g (dry weight) of cells, indicating the presence of a possible fourth osmolyte transporter.

ATP-Binding Cassette Transporters↗

PubMed2002

Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress.

Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.

Bifidobacterium↗