Dieter Weichart

Viability and activity in readily culturable bacteria: a review and discussion of the practical issues

In microbiology the terms ‘viability’ and ‘culturability’ are often equated. However, in recent years the apparently self-contradictory expression ‘viable-but-nonculturable’ (‘VBNC’) has been applied to cells with various and often poorly defined physiological attributes but which, nonetheless, could not be cultured by methods normally appropriate to the organism concerned. These attributes include apparent cell integrity, the possession of some form of measurable cellular activity and the apparent capacity to regain culturability. We review the evidence relating to putative VBNC cells and stress our view that most of the reports claiming a return to culturability have failed to exclude the regrowth of a limited number of cells which had never lost culturability. We argue that failure to differentiate clearly between use of the terms ‘viability’ and ‘culturability’ in an operational versus a conceptual sense is fuelling the current debate, and conclude with a number of proposals that are designed to help clarify the major issues involved. In particular, we suggest an alternative operational terminology that replaces ‘VBNC’ with expressions that are internally consistent.

How do non-differentiating bacteria adapt to starvation?

Non-differentiating bacteria adapt to starvation induced growth arrest by a complex turn-on/turn-off pattern of protein synthesis. This response shows distinct similarities with those of spore formation in differentiating organisms. A substantial amount of information on the non-growth biology of non-differentiating bacteria can be derived from studies onVibrio strains. One important result is that carbon rather than nitrogen or phosphorus starvation leads to the development of a starvation and stress resistant cell in these organisms. Hence, we have attempted to characterize the carbon starvation stimulon. By the use of two-dimensional gel electrophoresis of pulse-labelled cells and transposon mutagenesis, using reporter gene constructs, the identity and function of some members of the carbon starvation stimulon have been elucidated. Moreover, regulatory genes of the starvation response have been identified with these techniques. Current studies primarily address the identity and function of these genes. The role of transcript modification and stability for both long term persistence during starvation as well as the efficient recovery of cells which occurs upon nutrient addition is also addressed. It is suggested that an understanding of the functionality of the translational machinery is essential for the understanding of these adaptive pathways. This contribution also discusses the diversity of the differentiation-like response to starvation in different bacteria and whether a general starvation induced programme exists.

Stress resistance and recovery potential of culturable and viable but nonculturable cells of Vibrio vulnificus.

The estuarine, human-pathogenic bacterium Vibrio vulnificus responds to low temperature by the formation of viable but nonculturable (VBNC) cells, while starvation at moderate temperatures allows for maintenance of culturability of this organism. Recovery of cold-incubated populations of V. vulnificus was restricted to the culturable fraction in slide cultures and most probable number assays. These populations, however, gave between 1.1- and 8-fold higher c.f.u. counts on soft agar plates than on ordinary agar plates, indicating that a small and variable fraction of the cell population was injured rather than nonculturable. Thus, the population of cold-incubated cells is composed of culturable, injured and nonculturable cells, with the numbers of the culturable and injured cells rapidly decreasing during cold incubation. Recovery of nonculturable cells of the organism, however, could not be obtained by any combination of temperature and nutrient shifts in any of the assays. VBNC cells of the organism were assessed with regard to their persistence and stress resistance in comparison to growing and starved cells. The sonication resistance of VBNC cells was initially similar to that of growing cells, but increased during prolonged cold incubation. The final resistance of cold-incubated VBNC cells was equal to the markedly increased resistance of starving cells, which also displayed increased resistance against exposure to ethanol and mechanical stress. Our results indicate that in spite of the apparent absence of recovery under a wide range of laboratory conditions, VBNC cells of V. vulnificus undergo changes at low temperature which potentially allow them to persist for extended periods.

A model of yeast glycolysis based on a consistent kinetic characterisation of all its enzymes

We present an experimental and computational pipeline for the generation of kinetic models of metabolism, and demonstrate its application to glycolysis in Saccharomyces cerevisiae. Starting from an approximate mathematical model, we employ a “cycle of knowledge” strategy, identifying the steps with most control over flux. Kinetic parameters of the individual isoenzymes within these steps are measured experimentally under a standardised set of conditions. Experimental strategies are applied to establish a set of in vivo concentrations for isoenzymes and metabolites. The data are integrated into a mathematical model that is used to predict a new set of metabolite concentrations and reevaluate the control properties of the system. This bottom‐up modelling study reveals that control over the metabolic network most directly involved in yeast glycolysis is more widely distributed than previously thought.

Analysis of starvation conditions that allow for prolonged culturability of Vibrio vulnificus at low temperature

The response of the estuarine human pathogen Vibrio vulnificus to starvation for carbon, nitrogen or phosphorus, or all three nutrients simultaneously (multiple-nutrient), was examined with respect to the maintenance of culturability during incubation at low temperature. V. vulnificus showed similar survival patterns during starvation for the individual nutrients when kept at 24 degrees C. On the other hand, cultures prestarved at 24 degrees C and then shifted to 5 degrees C maintained culturability at low temperature in a starvation-condition-dependent manner. Carbon and multiple-nutrient starvation were indistinguishable in their ability to mediate maintenance of culturability in the cold. Prolonged starvation for phosphorus had a similar effect, but nitrogen starvation did not allow for maintenance of culturability. Extracellular factors produced during starvation were not observed to have an effect on the culturability of cells incubated at low temperature. Protein synthesis during starvation for individual nutrients was analysed by two-dimensional PAGE of pulse-labelled proteins. Carbon and multiple-nutrient starvation gave nearly identical protein induction patterns involving at least 34 proteins, indicating that carbon starvation determines both responses. Nitrogen starvation for 1 h induced 24 proteins, while phosphorus starvation induced a set of 10 proteins after 1 h and about 40 proteins after 18 h. It is suggested that starvation for carbon or phosphorus induces maintenance of culturability of V. vulnificus incubated at low temperature via the synthesis of distinct sets of starvation-specific proteins.

Estimation of Microbial Viability Using Flow Cytometry

For microorganisms in particular, viability is a term that is difficult to define and a state consequently difficult to measure. The traditional (and gold‐standard) usage equates viability and culturability (i.e., the ability to multiply), but the process of determining culturability is often too slow. Flow cytometry provides the opportunity to make rapid and quantitative measurements of dye uptake in large numbers of cells, and we can therefore exploit the flow cytometric approach to evaluate so‐called viability stains and to develop protocols for more routine assessments of microbial viability. This unit is primarily commentary, but several basic protocols have been included to ensure that users have a firm basis for attempting these reasonably difficult assays on traditional flow cytometer instruments. What is clear is that each assay must be carefully validated with the particular microorganism of interest before being applied in any research, clinical, or service form.

Analysis of NOD2-mediated proteome response to muramyl dipeptide in HEK293 cells.

NOD2, a cytosolic receptor for the bacterial proteoglycan fragment muramyl dipeptide (MDP), plays an important role in the recognition of intracellular pathogens. Variants in the bacterial sensor domain of NOD2 are genetically associated with an increased risk for the development of Crohn disease, a human chronic inflammatory bowel disease. In the present study, global protein expression changes after MDP stimulation were analyzed by two-dimensional PAGE of total protein extracts of human cultured cells stably transfected with expression constructs encoding for wild type NOD2 (NOD2WT) or the disease-associated NOD2 L1007fsinsC (NOD2SNP13) variant. Differentially regulated proteins were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) peptide mass fingerprinting and MALDI MS/MS. The limited overlap in the responses of the NOD2-overexpressing cell lines to MDP included a down-regulation of heat shock 70-kDa protein 4. A complex pro-inflammatory program regulated by NOD2WT that encompasses a regulation of key genes involved in protein folding, DNA repair, cellular redox homeostasis, and metabolism was observed both under normal growth conditions and after stimulation with MDP. By using the comparison of NOD2WT and disease-associated NOD2SNP13 variant, we have identified a proteomic signature pattern that may further our understanding of the influence of genetic variations in the NOD2 gene in the pathophysiology of chronic inflammatory bowel disease.

Systematic integration of experimental data and models in systems biology

BackgroundThe behaviour of biological systems can be deduced from their mathematical models. However, multiple sources of data in diverse forms are required in the construction of a model in order to define its components and their biochemical reactions, and corresponding parameters. Automating the assembly and use of systems biology models is dependent upon data integration processes involving the interoperation of data and analytical resources.ResultsTaverna workflows have been developed for the automated assembly of quantitative parameterised metabolic networks in the Systems Biology Markup Language (SBML). A SBML model is built in a systematic fashion by the workflows which starts with the construction of a qualitative network using data from a MIRIAM-compliant genome-scale model of yeast metabolism. This is followed by parameterisation of the SBML model with experimental data from two repositories, the SABIO-RK enzyme kinetics database and a database of quantitative experimental results. The models are then calibrated and simulated in workflows that call out to COPASIWS, the web service interface to the COPASI software application for analysing biochemical networks. These systems biology workflows were evaluated for their ability to construct a parameterised model of yeast glycolysis.ConclusionsDistributed information about metabolic reactions that have been described to MIRIAM standards enables the automated assembly of quantitative systems biology models of metabolic networks based on user-defined criteria. Such data integration processes can be implemented as Taverna workflows to provide a rapid overview of the components and their relationships within a biochemical system.

Characterization of an autostimulatory substance produced by Escherichia coli

The recovery of dilute populations of stationary phase cells of Escherichia coli was studied using an automatic growth analyser. The addition of 30% supernatant from 2-d-old stationary phase cells of the organism reproducibly shortened the apparent lag times by 22-57.5%, depending on the age of the inoculum. True lag times, as determined by colony counts, of stationary phase cells were reduced by supernatant addition by 41-62%. The growth-stimulating substance was characterized and partly purified from supernatants: the active material was shown to be dialysable, heat-stable, acid- and alkali-stable and protease-resistant. Extraction with ethyl acetate or ion-exchange resins was not successful, but the active material could be quantitatively extracted with ethanol after saturation with salt. It is concluded that the active substance is a small, non-proteinaceous, non-ionic organic molecule. Separation of extracts by HPLC indicated that the stimulatory substance is weakly hydrophobic and has retention times similar to those of uracil. So far, however, the exact chemical identity of the active substance has not been elucidated.

UNIT 11.3 Estimation of Microbial Viability Using Flow Cytometry

For microorganisms in particular, viability is a term that is difficult to define and a state consequently difficult to measure. The traditional (and gold‐standard) usage equates viability and culturability (i.e., the ability to multiply), but the process of determining culturability is often too slow. Flow cytometry provides the opportunity to make rapid and quantitative measurements of dye uptake in large numbers of cells, and we can therefore exploit the flow cytometric approach to evaluate so‐called viability stains and to develop protocols for more routine assessments of microbial viability. This unit is primarily commentary, but several basic protocols have been included to ensure that users have a firm basis for attempting these reasonably difficult assays on traditional flow cytometer instruments. What is clear is that each assay must be carefully validated with the particular microorganism of interest before being applied in any research, clinical, or service form.