Jan P. P. M. Smelt

Modelling the effect of sub(lethal) heat treatment of Bacillus subtilis spores on germination rate and outgrowth to exponentially growing vegetative cells

Spores of Bacillus subtilis were subjected to relatively mild heat treatments in distilled water and properties of these spores were studied. These spores had lost all or part of their dipicolinic acid (DPA) depending on the severity of the heat treatment. Even after relatively mild heat treatments these spore lost already a small but significant amount of DPA. When these spores were inoculated in nutrient medium-tryptone soy broth (TSA)-the non-lethally heated spores started to germinate. Results of classical optical density measurements showed that both phase darkening and subsequent outgrowth could be affected by sub-lethal heat. A study of single cells in TSB showed that lag times originating from exponentially growing cells followed a normal distribution, whereas lag times originating from spores followed a Weibull distribution. Besides classical optical density measurements were made to study the effect of previous heating on the kinetics of the first stages of germination. The germination kinetics could be described by the model as was proposed by Geeraerd et al. [Geeraerd, A.H., Herremans, C.H. and Van Impe, J.F., 2000. Structural model requirements to describe microbial inactivation during a mild heat treatment. International Journal of Food Microbiology 59, 185-209]. Two of the 4 parameters of the sigmoid model of Geeraerd were dependent on heating time and heating temperature, whereas the two other parameters were considered as independent of the heating conditions. Based on these observations, a secondary model could be developed that describes the combined effect of heating temperature and heating time on the kinetics of germination. To have more detailed information of the kinetics of germination samples incubated in TSB were tested at regular time intervals by flow cytometry. To that end the cells were stained with syto 9 to distinguish between the various germination stages. There was a qualitative agreement between the results of flow cytometry and those of optical density measurements, but there was a difference in quantitative terms. The results have shown that germination rate of spores is dependent on previous heating conditions both in the first stage when phase darkening occurs and also during the later stages of outgrowth when the phase dark spore develops to the vegetative cell.

Live cell imaging of germination and outgrowth of individual Bacillus subtilis spores; the effect of heat stress quantitatively analyzed with SporeTracker

Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.

Thermal Inactivation of Microorganisms

This paper serves as an overview of various aspects of thermal processing. Heat processing of foods has a long history and is still one of the most important preservation methods. To guarantee microbiological safety and stability, large safety margins are often applied in traditional heat processes. Because of the need for more fresh like foods, there is a need for milder preservation methods without compromising on safety and stability. The review deals with heat resistance data and mathematical models that describe heat inactivation. The effects of food composition are not yet fully clear and more knowledge of the cell physiology of the target microorganism could be of help in predicting the effects of food constituents. Finally, special attention has been paid to biological time temperature indicators to enable proper process calculations.

Effect of growth rate and selection pressure on rates of transfer of an antibiotic resistance plasmid between E. coli strains

Antibiotic resistance increases costs for health care and causes therapy failure. An important mechanism for spreading resistance is transfer of plasmids containing resistance genes and subsequent selection. Yet the factors that influence the rate of transfer are poorly known. Rates of plasmid transfer were measured in co-cultures in chemostats of a donor and a acceptor strain under various selective pressures. To document whether specific mutations in either plasmid or acceptor genome are associated with the plasmid transfer, whole genome sequencing was performed. The DM0133 TetR tetracycline resistance plasmid was transferred between Escherichia coli K-12 strains during co-culture at frequencies that seemed higher at increased growth rate. Modeling of the take-over of the culture by the transformed strain suggests that in reality more transfer events occurred at low growth rates. At moderate selection pressure due to an antibiotic concentration that still allowed growth, a maximum transfer frequency was determined of once per 10(11) cell divisions. In the absence of tetracycline or in the presence of high concentrations the frequency of transfer was sometimes zero, but otherwise reduced by at least a factor of 5. Whole genome sequencing showed that the plasmid was transferred without mutations, but two functional mutations in the genome of the recipient strain accompanied this transfer. Exposure to concentrations of antibiotics that fall within the mutant selection window stimulated transfer of the resistance plasmid most.

Challenges and advances in systems biology analysis of Bacillus spore physiology; molecular differences between an extreme heat resistant spore forming Bacillus subtilis food isolate and a laboratory strain

Bacterial spore formers are prime organisms of concern in the food industry. Spores from the genus Bacillus are extremely stress resistant, most notably exemplified by high thermotolerance. This sometimes allows surviving spores to germinate and grow out to vegetative cells causing food spoilage and possible intoxication. Similar issues though more pending toward spore toxigenicity are observed for the anaerobic Clostridia. The paper indicates the nature of stress resistance and highlights contemporary molecular approaches to analyze the mechanistic basis of it in Bacilli. A molecular comparison between a laboratory strain and a food borne isolate, very similar at the genomic level to the laboratory strain but generating extremely heat resistant spores, is discussed. The approaches cover genome-wide genotyping, proteomics and genome-wide expression analyses studies. The analyses aim at gathering sufficient molecular information to be able to put together an initial framework for dynamic modelling of spore germination and outgrowth behaviour. Such emerging models should be developed both at the population and at the single spore level. Tools and challenges in achieving the latter are succinctly discussed.

Intracellular pH Response to Weak Acid Stress in Individual Vegetative Bacillus subtilis Cells

ABSTRACT Intracellular pH (pHi) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pHi homeostasis. Unfortunately, accurate pHi quantification with existing methods is suboptimal, since measurements are averages across populations of cells, not taking into account interindividual heterogeneity. Yet, physiological heterogeneity in isogenic populations is well known to be responsible for differences in growth and division kinetics of cells in response to external stressors. To assess in this context the behavior of intracellular acidity, we have developed a robust method to quantify pHi at single-cell levels in Bacillus subtilis. Bacilli spoil food, cause disease, and are well known for their ability to form highly stress-resistant spores. Using an improved version of the genetically encoded ratiometric pHluorin (IpHluorin), we have quantified pHi in individual B. subtilis cells, cultured at an external pH of 6.4, in the absence or presence of weak acid stresses. In the presence of 3 mM potassium sorbate, a decrease in pHi and an increase in the generation time of growing cells were observed. Similar effects were observed when cells were stressed with 25 mM potassium acetate. Time-resolved analysis of individual bacteria in growing colonies shows that after a transient pH decrease, long-term pH evolution is highly cell dependent. The heterogeneity at the single-cell level shows the existence of subpopulations that might be more resistant and contribute to population survival. Our approach contributes to an understanding of pHi regulation in individual bacteria and may help scrutinizing effects of existing and novel food preservation strategies. IMPORTANCE This study shows how the physiological response to commonly used weak organic acid food preservatives, such as sorbic and acetic acids, can be measured at the single-cell level. These data are key to coupling often-observed single-cell heterogeneous growth behavior upon the addition of weak organic acid food preservatives. Generally, these data are gathered in the form of plate counting of samples incubated with the acids. Here, we visualize the underlying heterogeneity in cellular pH homeostasis, opening up avenues for mechanistic analyses of the heterogeneity in the weak acid stress response. Thus, microbial risk assessment can become more robust, widening the scope of use of these well-known weak organic acid food preservatives.

Erratum for Pandey et al., “Intracellular pH Response to Weak Acid Stress in Individual Vegetative Bacillus subtilis Cells”

Rachna Pandey,a Norbert O. E. Vischer,a Jan P. P. M. Smelt,a Johan W. A. van Beilen,a Alexander Ter Beek,a Winnok H. De Vos,b,c Stanley Brul,a Erik M. M. Mandersd Molecular Biology and Microbial Food Safety, SILS, University of Amsterdam, Amsterdam, The Netherlandsa; Department of Veterinary Sciences, Laboratory of Cell Biology and Histology, Antwerp University, Antwerp, Belgiumb; Department Molecular Biotechnology, Cell Systems and Imaging Group, Ghent University, Ghent, Belgiumc; Van Leeuwenhoek Centre for Advance Microscopy, SILS, University of Amsterdam, Amsterdam, The Netherlandsd

Inactiviation of microorganisms by high pressure

Microbial inactivation by high pressure is, in many respects, comparable to heat: the inactivation kinetics are the same and both heat and pressure can induce sublethal damage of the cells, reflected by a prolonged lag time. Bacterial cells are more sensitive to pressure and to heat at low pH and a low water activity gives the same protection for cells against pressure as it does against heat. We have shown that carbohydrates protect cells against pressure in the order trehalose > sucrose > glucose > fructose > glycerol. The membrane protective effect of these sugars is also in this order. There is also sufficient circumstantial evidence that the composition of the membrane correlates with resistance to pressure. The inactivation patterns of proteins and bacteria by pressure are quite similar and therefore it is reasonable to assume that inactivation of enzymes plays a role in pressure inactivation. We have therefore focused our attention on the membrane. After pressure treatment the intracellular pH could not be maintained at the same level as in untreated cells. This might be due to damage to the glycolytic pathway or inactivation of membrane bound enzymes, for example F0F1, ATPase. We have not yet investigated the effects of pressure on the glycolytic pathway. F0F1 ATPase was inactivated at high pressure. The latter observation suggests that membrane bound proteins would be involved in high pressure inactivation.