M. Ritz

Effects of high hydrostatic pressure on membrane proteins of Salmonella typhimurium

Salmonella typhimurium is a leading cause of foodborne diseases. Today high hydrostatic pressure treatments are considered as alternative methods of preservation. To select optimal conditions of treatment, we have to characterize the cell targets of pressure. In this study the action of pressure on the bacterial membrane proteins is analysed. The total membrane extract is obtained by lysis of cells separated by equilibrium density gradient centrifugation. Protein content is analysed by electrophoresis SDS-PAGE and visualised by silver stain. Electrophoretic profiles reveal the presence of three major outer membrane proteins and 12 minor proteins in control bacteria outer membranes. Outer membrane protein content is drastically modified after treatments. In some cases, except for the major proteins OmpA and LamB, other outer membrane proteins seem to totally disappear. LamB is more resistant to hyperbaric exposure when the pH of the media is acidic. This behaviour could be explained by a different conformation adopted by the LamB protein depending on the extracellular pH. This work allows us to define membrane proteins as a target of high hydrostatic pressure treatments. Knowledge of the behaviour of these bacterial membrane proteins subjected to pressure under different conditions (pH, temperature, a(w)...) could allow an increase in the efficiency of treatments.

Physiological effects of high hydrostatic pressure treatments on Listeria monocytogenes and Salmonella typhimurium

The effect of a high hydrostatic pressure treatment on the Gram‐positive Listeria monocytogenes strain Scott A and the Gram‐negative Salmonella typhimurium strain Mutton (ATCC13 311) has been determined in stationary phase cell suspensions. Pressure treatments were done at room temperature for 10 min in sodium citrate (pH 5·6) and sodium phosphate (pH 7·0) suspension buffers. Increasing pressure treatments resulted in an exponential decrease of cell counts. Salmonella typhimurium suspended at low pH was more sensitive to pressure treatments. Progressive morphological changes were evident with the pressure increase. Cell lysis only appeared with the highest pressure treatments. Cell volume was not affected by pressure treatment. A progressive decrease of δpH (pHin– pHout), intracellular potassium and ATP contents was demonstrated with the pressure increase. A parallel lowering of membrane potentials was measured.

Physiological damages of Listeria monocytogenes treated by high hydrostatic pressure

High hydrostatic pressure is a new food preservation technology known for its capacity to inactivate spoilage and pathogenic microorganisms. This study investigated the damages inflicted on Listeria monocytogenes cells treated by high pressure for 10 min at 400 MPa in pH 5.6 citrate buffer. Under these conditions, no cell growth occurred after 48 h on plate count agar. Scanning electron microscopy (SEM) revealed that cellular morphology was not really affected. Measuring propidium iodide (PI) staining followed by flow cytometry demonstrated that membrane integrity was damaged in a small part of the population, although the membrane potential evaluated by oxonol fluorescence or measured by analytical methods was reduced from - 86 to - 5 mV. These results for the first time showed that such combined methods as fluorescent dyes monitored by flow cytometry and physiological activity measurements provide valuable indications on cellular viability.

Survival of Campylobacter jejuni Strains from Different Origins Under Oxidative Stress Conditions: Effect of Temperature

Campylobacter jejuni is a microaerophilic pathogen but is able to survive oxidative stress conditions during its transmission to the human host. Strains of different origins (reference, poultry, or human clinical) were tested for survival under oxidative stress conditions. C. jejuni strains were grown in Mueller Hinton broth to obtain late exponential–phase cultures. Then they were exposed to 2 different stresses: (1) cultures were either plated on Columbia agar plates and exposed to atmospheric oxygen or (2) paraquat (a chemical oxidizing agent) was added to liquid cultures to reach a 500-μM concentration. Both of these experimental conditions were realized at 3 different temperatures: 4°C, 25°C, and 42°C. Results obtained with paraquat and atmospheric oxygen were similar. Surprisingly, C. jejuni was found to be very sensitive to oxidative stress at 42°C, which is its optimal growth temperature, whereas it was more resistant at 4°C. A strain effect was observed, but no relationship was found between the origin of the strains and level of resistance. High temperature (42°C) combined with oxidative stress allowed a rapid decrease in the C. jejuni population, whereas low temperature considerably decreased the effect of oxidative stress.

Determination of rpoA as the most suitable internal control to study stress response in C. jejuni by RT-qPCR and application to oxidative stress

Campylobacter jejuni represents one of the major causes of bacterial enteritis caused by food in humans. There are still mechanisms to be deciphered to better understand better its physiology and pathogenesis. Study of gene expression levels by RT-qPCR could be used, but to be accurate and reproducible, a good internal control has to be chosen. The aim of this study was to identify a highly stable housekeeping gene in Campylobacter jejuni that could constitute a good internal control to study gene expression variations between different growth phases or stress conditions. Expression levels of six different housekeeping genes (gyrA, ilvC, rpoA, slyD, thiC and rrs) were measured by RT-qPCR under different conditions (exponential phase, stationary phase, cold shock, cold shock+oxidative stress, oxidative stress). The rpoA gene was chosen as the best internal control. In a previous study, 9 proteins were identified as involved in oxidative stress response, among which 3 virulence factors. Expression levels of genes coding for these proteins was evaluated by RT-qPCR using rpoA as an internal control. The results obtained were concordant with what had been observed at the proteomic level, validating the methods used and confirming the hypothesis of a potential link between oxidative stress and virulence factors expression.

Role of the Cj1371 periplasmic protein and the Cj0355c two-component regulator in the Campylobacter jejuni NCTC 11168 response to oxidative stress caused by paraquat.

Campylobacter jejuni is a microaerophilic pathogen representing one of the major causes of bacterial enteritis in humans. The oxidative stress response after exposure to paraquat, a strong oxidising agent, was analysed by two-dimensional protein electrophoresis and Maldi-ToF mass spectrometry. Oxidative stress and redox-related proteins were overexpressed: FldA flavodoxin and a pyruvate-flavodoxin oxidoreductase encoded by cj1476c. No increase in SodB expression was observed. An additional quantitative RT-PCR analysis showed an increase in katA but not in sodB expression. However, the sodB mutant was very sensitive to paraquat, its basal expression level being essential for oxidative stress resistance. Proteins related to iron homeostasis (Cft and a non-haem iron protein encoded by cj0012c) and general stress response (FusA and MreB) were found overexpressed. Interestingly, a two-component regulator encoded by cj0355c was differentially expressed in the presence of paraquat and could play a role in induction of the C. jejuni oxidative stress response. Virulence factors (CadF, FlaA and a VacJ homolog encoded by cj1371) were also found overexpressed under oxidative stress conditions and a cj1371 mutant showed increased sensitivity to paraquat, suggesting that the Cj1371 periplasmic protein could play a role in C. jejuni oxidative stress resistance.

Effects of High Pressure, Subzero Temperature, and pH on Survival of Listeria monocytogenes in Buffer and Smoked Salmon

High pressure processing is a novel food preservation technology, applied for over 15 years in the food industry to inactivate spoilage and pathogenic microorganisms. Many studies have shown the differential resistance of bacterial cells to high pressure. Listeria monocytogenes is a bacterium able to grow at refrigerated temperature and to survive for a long time in minimally processed foods such as raw smoked fish. The freezing process does not cause significant decline of L. monocytogenes. The phase diagram of water under pressure permits a pressure treatment under subzero temperature, without the disadvantages of freezing for food quality. The aim of this study was to estimate if combined effects of pressure and subzero temperature could increase the destruction of L. monocytogenes in buffer and in smoked salmon. We investigated effects of high pressure processing (100, 150, and 200 MPa) combined with subzero temperatures (-10, -14, and -18 degrees C) and pH (7.0 and 4.5). Results showed that the most effective high-pressure treatment to inactivate L. monocytogenes was 200 MPa, -18 degrees C, and pH 4.5. The relevance of pressure holding time and the synergistic effect of pressure coupled with the subzero temperature to inactivate bacteria are highlighted. Modifications of physical properties (color and texture) were a lightening of color and an increase of toughness, which might be accepted by consumers, since safety is increased.

Impacts of treatment parameters on the inactivation of Campylobacter jejuni by high pressure: a statistical study of main effects and interactions

Aim:  The influence of environmental (temperature and pH) and biological (strain) parameters on the inactivation of Campylobacter jejuni by high hydrostatic pressure (HHP) was investigated.