G. Cebrián

Variation in resistance of natural isolates of Staphylococcus aureus to heat, pulsed electric field and ultrasound under pressure

Aims:  To study and compare the resistance of 15 Staphylococcus aureus isolates to heat, pulsed electric field (PEF) and ultrasound (UW) under pressure (manosonication, MS).

Development of stress resistance in Staphylococcus aureus after exposure to sublethal environmental conditions.

The ability of Staphylococcus aureus to develop stress resistance responses was investigated. Exponential growth phase cells of S. aureus CECT 4459 were exposed to sublethal conditions (acid and alkaline pH, hydrogen peroxide, and heat) and then the acquisition of resistance to acid (pH 2.5), alkali (pH 12.0), hydrogen peroxide (50mM), and heat (58 degrees C) was determined. Conditions resulting in the maximum development of homologous resistance (tolerance to the same stress), while preventing lethal effects in the population, were pH 4.5 (2h), pH 9.5 (30 min), 0.05 mM H(2)O(2) (30 min), and 45 degrees C (2h). Under these adaptation conditions, times for the first decimal reduction (TFDC) to a lethal treatment at acid pH, alkaline pH, hydrogen peroxide, and heat were increased by a factor of 1.6, 2, 2, and 6, respectively. The presence of chloramphenicol or rifampicin in the adaptation medium completely abolished the increase in homologous resistance to acid pH and to hydrogen peroxide. By contrast, the development of homologous resistance to alkaline pH resulted independently of the presence of either chloramphenicol or rifampicin. S. aureus heat resistance increased in the presence of the inhibitors during the heat shock, but only partially. In some cases, the exposure to a given stress induced cross-protection against other agents. Protective combinations of sublethal stress and lethal agents were: acid pH-heat, acid pH-hydrogen peroxide, alkaline pH-hydrogen peroxide, heat-acid pH, and heat-hydrogen peroxide. These combinations of agents applied sequentially should be avoided in food-processing environments.

Pulsed electric fields cause sublethal injuries in the outer membrane of Enterobacter sakazakii facilitating the antimicrobial activity of citral

Aims:  The objective was to evaluate the relation of sublethal injury in the outer membrane of Enterobacter sakazakii to the inactivating effect of the combination of pulsed electric fields (PEF) treatments and citral.

Inactivation of Cronobacter sakazakii by ultrasonic waves under pressure in buffer and foods

The objective of this research was to characterize the resistance of Cronobacter sakazakii to ultrasonic waves under pressure (manosonication, MS). The D(MS) value (decimal reduction time value) of C. sakazakii in standard conditions (35°C, 117 μm, 200 kPa, citrate-phosphate buffer pH 7.0) was 0.41 min. This value was higher than that of Yersinia enterocolitica (D(MS)=0.19 min) and lower than those of Salmonella enterica serovar Enteritidis (D(MS)=0.61 min), Listeria monocytogenes (D(MS)=0.86 min), and Enterococcus faecium (D(MS)=1.2 min). Strain studied (ATCC 29544, NCTC 8155, 9238, and 9529), growth temperature (10, 20, 30, and 37°C), and pH of the treatment media (4.0, 5.0, 6.0, and 7.0) did not significantly change C. sakazakii MS resistance. Conversely, entry into stationary growth phase, decreasing water activity of the treatment media (0.98, 0.96, and 0.94), and treatment in food products (apple and orange juices, chicken and vegetable soups, and rehydrated powdered milk) resulted in up to a 1.6-, 3.9-, and 2.5-fold maximum change in D(MS) values, respectively. Whereas an exponential relationship between the amplitude of ultrasonic waves and D(MS) values was found, the relationship between static pressure and D(MS) values was better described by a quadratic equation. The energy transferred into the medium determined the lethality of the ultrasonic waves regardless of the combination of pressure (0, 50, 100, 200 and 300 kPa) and amplitude (34, 62, 90, 117 and 145 μm) applied. There was an exponential relationship between D(MS) values and the power input: an increase of 134 W increased the inactivation rate ten times regardless of the treatment medium. No C. sakazakii cells with sublethally injured cytoplasmic membrane or with sublethal oxidative damage occurred after MS treatments, but the results indicated that damage to the outer membrane preceded microbial death.

Role of the alternative sigma factor σB on Staphylococcus aureus resistance to stresses of relevance to food preservation.

Aims:  To examine the role of the alternative general stress sigma factor σB on the resistance of Staphylococcus aureus to stresses of relevance to food preservation, with special emphasis on emerging technologies such as pulsed electric fields (PEF) and high hydrostatic pressure (HHP).

Inactivation of Cronobacter sakazakii by manothermosonication in buffer and milk.

The inactivation of Cronobacter sakazakii by heat and ultrasound treatments under pressure at different temperatures [manosonication (MS) and manothermosonication (MTS)] was studied in citrate-phosphate pH 7.0 buffer and rehydrated powdered milk. The inactivation rate was an exponential function of the treatment time for MS/MTS treatments (35-68 °C; 200 kPa of pressure; 117 μm of amplitude of ultrasonic waves) in both media, and for thermal treatments alone when buffer was used as heating media. Survival curves of C. sakazakii during heating in milk had a concave downward profile. Up to 50 °C, the lethality of ultrasound under pressure treatments was independent of the treatment temperature in both media. At temperatures greater than 64 °C in buffer and 68 °C in milk, the inactivating effect of MTS was equivalent to that of the thermal treatments alone at the same temperature. Between 50 and 64 ºC for buffer and 50 and 68 °C for milk, the lethality of MTS was the result of a synergistic effect, where the total lethal effect was higher than the lethal effect of heat added to that of ultrasound under pressure at room temperature. The maximum synergism was found at 60 °C in buffer and at 56 °C in milk. A heat treatment of 12 min (60 °C) or 4 min of an ultrasound under pressure at room temperature treatment (35 °C; 200 kPa; 117 μm) would be necessary to guarantee the death of 99.99% of C. sakazakii cells suspended in milk. The same level of C. sakazakii inactivation can be achieved with 1.8 min of a MTS treatment (60 °C; 200 kPa; 117 μm). Damaged cells were detected after heat treatments and after ultrasound under pressure treatments at lethal but not at non-lethal temperatures.

Induced thermotolerance under nonisothermal treatments of a heat sensitive and a resistant strain of Staphylococcus aureus in media of different pH

Aims:  The aim was to assess the induced thermotolerance under nonisothermal treatments of two strains of Staphylococcus aureus in media of different pH.

Development of resistance in Cronobacter sakazakii ATCC 29544 to thermal and nonthermal processes after exposure to stressing environmental conditions

Aims:  The objective was to study the response of Cronobacter sakazakii ATCC 29544 cells to heat, pulsed electric fields (PEF), ultrasound under pressure (Manosonication, MS) and ultraviolet light (UV‐C) treatments after exposure to different sublethal stresses that may be encountered in food‐processing environments.

Resistance of Escherichia coli grown at different temperatures to various environmental stresses.

Aims:  To study the influence of growth temperature on the resistance of Escherichia coli to three agents of different nature: heat, pulsed electric field (PEF) and hydrogen peroxide.

Manothermosonication for Microbial Inactivation

Ultrasound is one of the new technologies of microbial inactivation that has been suggested as an alternative to heat treatments. Despite the improvement of current ultrasound generators some data indicate that the germ-killing efficacy of the process is relatively low under atmospheric pressure and room temperature. Therefore most investigators have tried to improve the efficacy of the process, either by increasing cavitation intensity or by designing combined processes to enhance the lethal effect. This chapter reviews the accumulated knowledge in the last 15 years concerning the microbial lethal efficacy of ultrasonic waves under pressure at room temperatures (manosonication, MS) as well as at mild temperatures (manothermosonication, MTS). The chapter focuses on the microbial MS/MTS resistance and inactivation kinetics, on the effect of physical parameters on the lethality of the treatment and on its control. The mechanisms of action and the possibilities to design combined processes are also discussed.