Proteomics and microscopy tools for the study of antimicrobial resistance and germination mechanisms of bacterial spores.

Bacterial spores are ubiquitous in nature and can withstand both chemical and physical stresses. Spores can survive food preservation processes and upon outgrowth cause food spoilage as well as safety risks. The heterogeneous germination and outgrowth behavior of isogenic spore populations exacerbates this risk. A major unknown factor of spores is likely to be the inherently heterogeneous spore protein composition. The proteomics methods discussed here help in broadening the knowledge about spore structure and identification of putative target proteins from spores of different spore formers. Approaches to synchronize Bacillus subtilis spore formation, and to analyze spore proteins as well as the physiology of spore germination and outgrowth are also discussed. Live-imaging and fluorescence microscopy techniques discussed here allow analysis, at single cell level, of the 'germinosome', the process of spore germination itself, spore outgrowth and the spore intracellular pH dynamics. For the latter, a recently published improved pHluorin (IpHluorin) under control of the ptsG promoter is applicable. While the data obtained from such tools offers novel insight in the mechanisms of bacterial spore awakening, it may also be used to probe candidate antimicrobial compounds for inhibitory effects on spore germination and strengthen microbial risk assessment.

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.

Endovascular coiling of a mycotic external carotid artery pseudoaneurysm following pharyngolaryngectomy with a free jejunal graft.

OBJECTIVES: (1) To highlight the significance of carotid artery pseudoaneurysm as a rare complication following neck dissection, and (2) to suggest endovascular coiling as management, in the presence of infection, previous radiotherapy and a grafted blood supply. CASE REPORT: A 66-year-old man diagnosed with squamous cell carcinoma of the hypopharynx and upper oesophagus underwent pharyngolaryngectomy with reconstruction of a neo-pharynx using a free jejunal graft. The patient had previously received radiotherapy for a soft palate squamous cell carcinoma. Two months after surgery, computed tomography demonstrated a bilobed pseudoaneurysm of the left external carotid artery just distal to the arterial branch supplying the jejunal graft. This mycotic pseudoaneurysm was successfully treated with endovascular coiling, while maintaining the patency of the superior thyroid artery supplying the jejunal graft anastomosis. CONCLUSION: In this patient, endovascular coiling of the external carotid artery was considered to be the only definitive treatment for a life-threatening mycotic pseudoaneurysm.

Laparoscopic removal of a double gallbladder for cholelithiasis: a case report.

We describe the findings of symptomatic cholelithiasis in the double gallbladder of a 75-year-old woman, which was successfully removed laparoscopically. This report highlights the importance of this condition and the effectiveness of magnetic resonance cholangiopancreatography in defining abnormal gallbladder anatomy.

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.

Modelling and optimization of inactivation of Lactobacillus plantarum by pulsed electric field treatment.

AIMS: The effect of critical pulsed electric field (PEF) process parameters, such as electric field strength, pulse length and number of pulses, on inactivation of Lactobacillus plantarum was investigated. METHODS AND RESULTS: Experiments were performed in a pH 4.5 sodium phosphate buffer having a conductivity of 0.1 S m-1, using a laboratory-scale continuous PEF apparatus with a co-linear treatment chamber. An inactivation model was developed as a function of field strength, pulse length and number of pulses. Based on this inactivation model, the conditions for a PEF treatment were optimized with respect to the minimum energy required to obtain a certain level of inactivation. It was shown that the least efficient process parameter in the range investigated was the number of pulses. The most efficient way to optimize inactivation of Lact. plantarum was to increase the field strength up to 25.7 kV cm-1, at the shortest pulse length investigated, 0.85 micros, and using a minimum number of pulses. The highest inactivation of Lact. plantarum at the lowest energy costs is obtained by using the equation: E=26.7tau0.23, in which E is the field strength and tau the pulse length. An optimum is reached by substituting tau with 5.1. CONCLUSIONS: This study demonstrates that the correct choice of parameters, as predicted by the model described here, can considerably improve the PEF process. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge gained in this study improves the understanding of the limitations and opportunities of the PEF process. Consequently, the advantage of the PEF process as a new option for non-thermal decontamination can be better utilized.

Rate of bentazone transformation in four layers of a humic sandy soil profile with fluctuating water table.

The rate of transformation of a pesticide as a function of the depth in the soil is needed as an input into computations on the risk of residues leaching to groundwater. The herbicide bentazone was incubated at 15 degrees C in soil materials derived from four layers at depths of up to 2.5 m in a humic sandy soil profile with a fluctuating water table (0.8 to 1.4 m), while simulating the redox conditions existing in the field. Gamma-irradiation experiments indicated that bentazone is mainly transformed by microbial activity in the soil. The rate constant for transformation was highest in the humic sandy top layer; it decreased with depth in the sandy vadose subsoil. However, material from the top of the phreatic aquifer had a higher rate constant than that from the layers just above. The presence of fossil organic material in the fluviatile water-saturated sediment probably stimulated microbial activity and bentazone transformation. The changes in the transformation rate constant with depth showed the same trend as those in some soil factors, viz organic carbon content, water-extractable phosphorus and microbial density as measured by fluorescence counts. However, the (low) concentration of dissolved organic carbon (DOC) in the top of the aquifer did not fit the trend. The rate constant for bentazone transformation in the layers was higher at lower initial contents of the herbicide.

An appraisal of methods for measurement of pesticide transformation in the groundwater zone.

Laboratory and field studies show that pesticides may be transformed in the groundwater zone. Possible reaction mechanisms are chemical hydrolysis, catalytic reduction and aerobic or anaerobic microbial transformation. Transformation in the groundwater zone can be an important element in the advanced evaluation of the potential risk arising from a pesticide in the public drinking water supply. However, rate and pathway of transformation can show large differences, depending on the bio-geochemical conditions in the groundwater zone. Knowledge of the reaction mechanisms and the effect of aquifer conditions would allow vulnerable and low-vulnerable application areas for a pesticide to be delimited. An outline is given of possible approaches to quantifying these transformation processes and using the results in registration procedures, especially in the EU and its member states. Furthermore, areas where there is need for continued research and better understanding are highlighted.

Effects of pulsed electric fields on inactivation kinetics of Listeria innocua.

The effects of pulsed electric field (PEF) treatment and processing factors on the inactivation kinetics of Listeria innocua NCTC 11289 were investigated by using a pilot plant PEF unit with a flow rate of 200 liters/h. The electric field strength, pulse length, number of pulses, and inlet temperature were the most significant process factors influencing the inactivation kinetics. Product factors (pH and conductivity) also influenced the inactivation kinetics. In phosphate buffer at pH 4.0 and 0.5 S/m at 40 degrees C, a 3. 0-V/microm PEF treatment at an inlet temperature of 40 degrees C resulted in > or = 6.3 log inactivation of strain NCTC 11289 at 49.5 degrees C. A synergistic effect between temperature and PEF inactivation was also observed. The inactivation obtained with PEF was compared to the inactivation obtained with heat. We found that heat inactivation was less effective than PEF inactivation under similar time and temperature conditions. L. innocua cells which were incubated for a prolonged time in the stationary phase were more resistant to the PEF treatment, indicating that the physiological state of the microorganism plays a role in inactivation by PEF. Sublethal injury of cells was observed after PEF treatment, and the injury was more severe when the level of treatment was increased. Overall, our results indicate that it may be possible to use PEF in future applications in order to produce safe products.

Effects of High Pressure on Inactivation Kinetics and Events Related to Proton Efflux in Lactobacillus plantarum.

Knowledge of the mechanism of pressure-induced inactivation of microorganisms could be helpful in defining an effective, relatively mild pressure treatment as a means of decontamination, especially in combination with other physical treatments or antimicrobial agents. We have studied the effect of high pressure on Lactobacillus plantarum grown at pH 5.0 and 7.0. The classical inactivation kinetics were compared with a number of events related to the acid-base physiology of the cell, i.e., activity of F(0)F(1) ATPase, intracellular pH, acid efflux, and intracellular ATP pool. Cells grown at pH 5.0 were more resistant to pressures of 250 MPa than were cells grown at pH 7.0. This difference in resistance may be explained by a higher F(0)F(1) ATPase activity, better ability to maintain a DeltapH, or a higher acid efflux of the cells grown at pH 5.0. After pressure treatment at 250 MPa, the F(0)F(1) ATPase activity was decreased, the ability to maintain a DeltapH was reduced, and the acid efflux was impaired. The ATP pool increased initially after mild pressure treatment and finally decreased after prolonged treatment. The observations on acid efflux and the ATP pool suggest that the glycolysis is affected by high pressure later than is the F(0)F(1) ATPase activity. Although functions related to the membrane-bound ATPase activity were impaired, no morphological changes of the membrane could be observed.

Adsorption, degradation and leaching of pirimicarb in orchard soils.

This paper shows the results of experiments (adsorption, degradation and mobility) carried out to investigate the risk of pirimicarb leaching to groundwater, after its application to integrated fruit growing systems. Soil batches were collected from experimental fruit farms in Numansdorp and Zeewolde (The Netherlands). The distribution coefficients and the degradation rate coefficients for Numansdorp and Zeewolde soils, respectively, show a higher adsorption and degradation rate in the Zeewolde soil than in the Numansdorp soil. By leaching of pirimicarb in two soil columns, it was found that pirimicard mobility is lower in the Zeewolde soil columns than in the Numansdorp ones but, in both cases, it is much lower than it would be expected from their adsorption coefficients. Pirimicarb is detected in the first effluents, however, the highest concentration remained in the upper 10 cm of the soil columns. A computation model simulating uniform water flow and instantaneous adsorption desorption equilibrium is not suitable to describe the leaching of pirimicarb in structured loamy soils at relatively high water flow-rates.

A proposal for using previous experience in designing microbiological sampling plans based on variables.

A microbiological sampling system on the basis of variables is presented which requires only small numbers of replicates (n greater than or equal to 2). The system uses previous data on standard deviations of numbers of micro-organisms in batches and is particularly useful for in-plant situations. The discriminating power of the system is comparable to that of the current International Committee on Microbiological Specifications for Foods sampling schemes but uses more replicates.

Identification and grouping of Clostridium botulinum strains by numerical analysis of their electrophoretic protein patterns.

Strains of Clostridium botulinum type A, type E and both non-proteolytic and proteolytic types B and F were characterized by their electrophoretic protein patterns. As the protein pattern changes during sporulation, special attention was paid to the prevention of sporulation by selecting an appropriate medium (Strasdine's medium plus 1% w/v glucose) and a scheme of repeated subculturing. Ribosomal proteins, evolutionarily conservative and hence relatively similar in all types of bacteria, were removed to optimize the resolving power of the electrophoretic technique. Protein patterns were compared by computing correlation coefficients of normalized densitometric tracings. The method is highly reproducible and its resolving power is high: all protein patterns found were specific. The strains tested fall into two main groups: the proteolytic and the non-proteolytic cluster. Type A strains form a separate subgroup within the proteolytic cluster, the same applies to type E strains within the non-proteolytic group. Although time-consuming for spore-forming bacteria, this method is, to our knowledge, the only technique that recognizes individual strains of Cl. botulinum. For non-spore-forming micro-organisms the method is certainly much simpler and hence even more valuable.

Clostridium botulinum can grow and form toxin at pH values lower than 4.6.

It is generally accepted that in Clostridium botulinum both growth and toxin formation are completely inhibited at pH values below 4.6. This critical pH value has been confirmed by many investigators using food as substrate or culture media. Occasionally growth of C. botulinum and toxin formation at pH values lower than 4.6 have been reported. In these cases the authors ascribed the unexpected outgrowth and toxin formation to local pH differences in inhomogeneous media and growth of C. botulinum before pH equilibration, or to the fact that fungi created microenvironments within or adjacent to the mycelial mat, where the pH was higher than 4.6 as was demonstrated by Odlaug and Pflug. We show here that the general assumption that C. botulinum does not grow below pH 4.6 is incorrect. We have observed that growth and toxin formation by C. botulinum can take place in homogeneous protein rich substrates (containing 3% or more soya or milk protein) at pH values lower than 4.6.