Comparison between the resistance of benzalkonium chloride-adapted and -nonadapted biofilms of Listeria monocyogenes to modified atmosphere packaging and nisin once transferred to mussels.

Benzalkonium chloride-adapted and -nonadapted Listeria monocytogenes biofilm cells were transferred by contact to cooked or live mussels and packed in rich CO(2) and O(2), respectively. The viabilities of transferred cells during storage of these packed samples at 2.5 °C were compared. In addition, in cooked mussels the combined effect of CO(2) and nisin against the survival of L. monocytogenes was also studied by using a first-order factorial design. The results obtained demonstrated that biofilms formed by benzalkonium chloride-adapted L. monocytogenes cells could be more resistant to the application of modified atmospheres rich in CO(2) and nisin once they have been transferred to cooked mussels by contact (simulating cross-contamination). This implies an increase in the risk associated with the presence of these cells in food processing plants. Significant empirical equations obtained after 7, 11, and 20 days showed an inhibitory effect of CO(2) and nisin against L. monocytogenes. However, a significant positive interaction between both variables highlights an incompatibility between CO(2) and nisin at high concentrations. Results also demonstrated that L. monocytogenes could persist after cross-contamination during the processing of live mussels, so L. monocytogenes is of concern as a contaminant in live mussels packaged in high-O(2) atmospheres.

Resistance to benzalkonium chloride, peracetic acid and nisin during formation of mature biofilms by Listeria monocytogenes.

Increase of resistance to the application of benzalkonium chloride (BAC), peracetic acid (PA) and nisin during biofilm formation at 25 °C by three strains of Listeria monocytogenes (CECT 911, CECT 4032, CECT 5873 and BAC-adapted CECT 5873) in different scenarios was compared. For this purpose, resistance after 4 and 11-days of biofilm formation was quantified in terms of lethal dose 90% values (LD(90)), determined according with a dose-response logistic mathematical model. Microscopic analyses after 4 and 11-days of L. monocytogenes biofilm formation were also carried out. Results demonstrated a relation between the microscopic structure and the resistance to the assayed biocides in matured biofilms. The worst cases being biofilms formed by the strain 4032 (in both stainless steel and polypropylene), which showed a complex "cloud-type" structure that correlates with the highest resistance of this strain against the three biocides during biofilm maturation. However, that increase in resistance and complexity appeared not to be dependent on initial bacterial adherence, thus indicating mature biofilms rather than planctonic cells or early-stage biofilms must be considered when disinfection protocols have to be optimized. PA seemed to be the most effective of the three disinfectants used for biofilms. We hypothesized both its high oxidizing capacity and low molecular size could suppose an advantage for its penetration inside the biofilm. We also demonstrated that organic material counteract with the biocides, thus indicating the importance of improving cleaning protocols. Finally, by comparing strains 5873 and 5873 adapted to BAC, several adaptative cross-responses between BAC and nisin or peracetic acid were identified.

A proteomic approach to cold acclimation of Staphylococcus aureus CECT 976 grown at room and human body temperatures.

Staphylococcus aureus is an important pathogenic microorganism that has been associated with serious infection problems in different fields, from food to clinic. In the present study, we have taken into account that the main reservoirs of this microorganism are the human body and some parts of food processing plants, which have normal temperatures of around 37 and 25°C, respectively. It can be expected that S. aureus must acclimate its metabolism to colder temperatures before growing in food matrices. Since temperature abuse for foods occurs at approximately 12°C, it is expected that S. aureus must acclimate its metabolism to colder temperatures before growing in food. For this reason, we have performed a proteomic comparison between exponential- and stationary-phase cultures of S. aureus CECT 976 acclimated to 12°C after growing at 25°C or 37°C. The analysis led to the identification of two different protein patterns associated with cold acclimation, denominated pattern A and pattern B. The first was characteristic of cultures at stationary phase of growth, grown at 25°C and acclimated to 12°C. The second appeared in the rest of experimental cases. Pattern A was distinguished by the presence of glycolytic proteins, whereas pattern B was differentiated by the presence of general stress and regulatory proteins. Pattern A was related through physiological experiments with a cross-resistance to acid pH, whereas pattern B conferred resistance to nisin. This prompted us to conclude that both molecular strategies could be valid, in vivo, for the process of acclimation of S. aureus to cold temperatures.

Application of nisin and pediocin against resistance and germination of Bacillus spores in sous vide products.

Sous vide and other mild preservation techniques are increasingly demanded by consumers. However, spores often will survive in minimally processed foods, causing both spoilage and safety problems. The main objective of the present work was to solve an industrial spoilage problem associated with two sous vide products: mushrooms and shellfish salad. Bacillus subtilis and Bacillus licheniformis predominated as the most heat-resistant organisms isolated from mushrooms and shellfish salad, respectively. The combined effects of nisin and pediocin against resistance and germination of both Bacillus species were described by empirical equations. Whereas nisin was more effective for decreasing thermal resistance of B. subtilis spores, pediocin was more effective against B. licheniformis. However, a significant positive interaction between both biopeptides for decreasing the proportion of vegetative cells resulting from thermoresistant spores was demonstrated in later experiments, thus indicating the increased efficacy of applying high concentrations of both bacteriocins. This efficacy was further demonstrated in additional challenge studies carried out at 15 degrees C in the two sous vide products: mushrooms and shellfish salad. Whereas no vegetative cells were detected after 90 days in the presence of bacteriocins, almost 100% of the population in nontreated samples of mushrooms and shellfish salad was in the vegetative state after 17 and 43 days of storage at 15 degrees C, respectively.

Adhesion and detachment kinetics of several strains of Staphylococcus aureus subsp. aureus under three different experimental conditions.

The kinetics of adhesion of five Staphylococcus aureus subsp. aureus strains (CECT 976, 4459, 4465, 4466 and 5191) to polypropylene at 25 degrees C in the absence of nutrients (PBS medium) were initially compared. Those strains with the highest (CECT 4459) and the lowest (CECT 976) adhesion levels were selected for further studying the effects of a nutrient-rich adhesion-promoting medium (TSB plus 1% glucose-TSBG) as well as of a conditioning film consisting of dried mussel cooking juices (MCJ) on adhesion to and detachment from polypropylene surfaces. Adhesion kinetics were properly described by an empirical model in the absence of conditioning film. The maximum adhesion level was much higher in the presence of TSBG than in PBS, decreasing sharply in both cases after 10-15 h. In contrast, adhesion increased exponentially during 25 h in the presence of dried MCJ. Clear differences were thus found in different media, and it suggests that cleaning strategies should vary under different conditions. The comparison of the adhesion strengths under the different experimental conditions showed that the persistence was highest when biofilms were formed on MCJ, which indicates that cells would remain longer as a source of cross-contamination. Some biofilms were examined by electronic microscopy, and different structures were observed under the different experimental conditions. It is concluded that the study of biofilm formation by S. aureus is necessary to establish efficient control systems in the food industry.