Quantitative image analysis to characterize the dynamics of Listeria monocytogenes biofilms.

This work shows that the combination of two-dimensional (2D) and three-dimensional (3D) analyses of images acquired by confocal laser scanning microscopy facilitates the quantitative spatiotemporal characterization of architectures formed by Listeria monocytogenes biofilms. In particular, the analysis of structural features such as maximum thickness, biovolume, areal porosity and maximum diffusion distance allowed elucidating differences in biofilm formation of three L. monocytogenes strains (L1A1, CECT5873 and CECT4032). The analysis showed a common sequence for all strains. In the first phase, independent clusters evolve to interconnected clusters and honeycomb-like structures. Flat biofilms characterized the second phase. The structures disappear in the third phase. Nevertheless, the duration of the phases differed from strain to strain. L1A1 strain exhibited the slowest dynamics and the thickest biofilms while the strain CECT4032 presented the faster dynamics and the thinnest biofilms. Also, the number of dead cells varies significantly from strain to strain. From the results of the analysis, it can be concluded that 2D parameters are critical to differentiating morphological features while 3D parameters ease the interpretation and comparative study of the different phases during the life cycle of biofilms.

Numerical spatio-temporal characterization of Listeria monocytogenes biofilms.

As the structure of biofilms plays a key role in their resistance and persistence, this work presents for the first time the numerical characterization of the temporal evolution of biofilm structures formed by three Listeria monocytogenes strains on two types of stainless-steel supports, AISI 304 SS No. 2B and AISI 316 SS No. 2R. Counting methods, motility tests, fluorescence microscopy and image analysis were combined to study the dynamic evolution of biofilm formation and structure. Image analysis was performed with several well-known parameters as well as a newly defined parameter to quantify spatio-temporal distribution. The results confirm the interstrain variability of L. monocytogenes species regarding biofilm structure and structure evolution. Two types of biofilm were observed: homogeneous or flat and heterogeneous or clustered. Differences in clusters and in attachment and detachment processes were due mainly to the topography and composition of the two surfaces although an effect due to motility was also found.

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.

Effects of mussel processing soils on the adherence of Listeria monocytogenes to polypropylene and stainless steel.

A comparative study of adhesion kinetics of L. monocytogenes (strains CECT 5873, CECT 936, CECT 911, and CECT 4032, representing serotypes 1/2a, 1/2b, 1/2c, and 4b, respectively) to polypropylene (PP) and stainless steel (SS) under two surface contamination conditions in plants processing cooked mussel was carried out. The conditions were either (i) contamination of clean surfaces with mussel cooking juice carrying L. monocytogenes or (ii) contamination with L. monocytogenes after soiling with mussel cooking juice, i.e., conditioning film (CF). The kinetics of adhesion were successfully described by a modified logistic model. Adhesion to PP was higher than to SS in all strains, except CECT 5873. Adhesion was initially higher in the presence of CF, but numbers of adherent cells decreased sharply in the late phase of study in three of eight cases as a result of cell detachment. Combinations of strain, surface material, and surface conditioning where adhesion was most enhanced were defined as the worst-case scenarios (CECT 911-PP, 4032-PP-CF, 5873-SS, and 4032-SS-CF). Subsequently, adhesion in worst-case scenarios was compared with a similar contamination event taking place in plants processing live mussels, using intervalvar water of mussel as a food residue matrix. Adhesion levels were higher in intervalvar water than in cooking juice, especially in both cases with no CF; this was attributed to more space available for adhesion or to physicochemical conditions enhancing cells to adhere.

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.

Survival of lactic acid bacteria in seawater: a factorial study.

A feasibility study of lactic bacteria as potential probiotics in larval cultures of marine fish was performed by investigating the survival of five strains of lactic bacteria in seawater by readily standardized procedures at different temperatures and salinities. These conditions were chosen in such a way that their combinations define a complete first-order factorial design. Depending on the strain and the ambient conditions, the survival adhered to first-order kinetics in some cases, and to the Gompertz equation in others. The half lives (t0.5) calculated from these models were subsequently introduced as responses to the factorial designs, estimating the coefficients of empirical equations that describe the group effect of temperature and salinity on t0.5. Simply additive effects were found in two cases, a negative first-order interaction in another case, while another two required second-order models.

Apparent antifungal activity of several lactic acid bacteria against Penicillium discolor is due to acetic acid in the medium.

Fifty-six dairy bacteria belonging to the genera Lactococcus, Lactobacillus, Pediococcus, Propionibacterium, Streptococcus, Enterococcus, Leuconostoc, and Brevibacterium were screened for antifungal activity against four species of fungi relevant to the cheese industry (Penicillium discolor, Penicillium commune, Penicillium roqueforti, and Aspergillus vesicolor). Most of the active strains belonged to the genus Lactobacillus, whereas Penicillium discolor was found to be the most sensitive of the four fungi investigated. Further studies on P. discolor showed antifungal activity only below pH 5. This effect of pH suggests that organic acids present in the culture could be involved in the detected activity. Determination of acid composition revealed lactic acid production for active dairy strains and the presence of acetic acid in active as well as inactive strains. It was demonstrated that the undissociated acetic acid originates from the bacterial growth medium. The synergistic effect of the acetic acid present and the lactic acid produced was likely the main factor responsible for the antifungal properties of the selected bacteria. These results could explain some discrepancies in reports of the antifungal properties of lactic acid bacteria, since the role of acetic acid has not been considered in previous studies.

An empirical model for describing the effects of nitrogen sources on nisin production.

AIMS: A study on the effects of tryptone and yeast extract on nisin production by Lactococcus lactis was carried out using a second order rotatable factorial design. METHODS AND RESULTS: The results show that both ingredients increased nisin production, although a small decrease in nisin levels was noted at high tryptone concentrations. In view of the low vitamin content of tryptone, the amino acids present in both tryptone and yeast extract may be responsible for these observations. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The observed response suggests that maintaining tryptone concentrations in the centre of the domain and increasing the concentration of yeast extract would be ideal conditions for nisin production. However, when the economic aspects are considered, it appears that low concentrations of yeast extract and reasonably high concentrations of tryptone are optimal.

Joint effect of nisin, CO2, and EDTA on the survival of Pseudomonas aeruginosa and Enterococcus faecium in a food model system.

A study on the joint effect of either nisin or Nisaplin, headspace CO2 levels, and EDTA on the survival of Pseudomonas aeruginosa and Enterococcus faecium was carried out in a water-soluble fish muscle extract at 3 degrees C using a second-order rotatable factorial design. E. faecium was completely deactivated by all processing after 2 days of storage. In contrast, P. aeruginosa was much less susceptible to treatments, and cell death was satisfactorily described by two models. Nisin increased cell death, whereas Nisaplin (commercial form of nisin) was not suitable, as it caused undesirable interference, presumably due to its co-compounds. Interactions between Nisaplin or nisin and either EDTA or CO2 were found to be nonstatistically significant. Factors that could account for this unexpected lack of synergism are discussed. However, a statistically significant positive interaction was found between CO2 and EDTA. This finding could allow CO2 levels to be decreased and hence to reduce the main disadvantages of CO2 application, namely, exudation and acidification.

Dose-response relationships. A model for describing interactions, and its application to the combined effect of nisin and lactic acid on Leuconostoc mesenteroides.

As well as producing bacteriocins, many lactic bacteria produce other potentially toxic compounds or growth inhibitors, especially lactic acid, which may interfere in the assays commonly used to quantify these peptides. A systematic set of modifications is proposed which, when applied to the logistical equation, enable it to describe the combined (but not additive) effects of two or more active principles. The general model thus derived is applied to the interaction of nisin and lactic acid on Leuconostoc mesenteroides.

A method for bacteriocin quantification.

Different aspects of the most commonly used assay methods in the study of bacteriocins were examined. The conditions under which extraction and incubation (including exposure time) take place were analysed, and several different formal models that are usually employed to calculate ID50 were compared. As an alternative designed to overcome the problems which characterize the response of micro-organisms that are sensitive to bacteriocins, an operative procedure in a liquid medium and a modified re-parameterized logistic equation is proposed. When applied to the inhibition of Leuconostoc mesenteroides by nisin, the model allows an optimal experimental procedure to be defined.

Effect of various cryostabilizers on the production and reactivity of formaldehyde in frozen-stored minced blue whiting muscle.

The production of formaldehyde in frozen-stored minced blue whiting muscle was described by a rectangular hyperbolic model, and the effectiveness of each cryostabilizer is discussed in terms of its parameters. The maltodextrins assayed noticeably inhibited formaldehyde production, this effect being greater at -20 degrees C than at -10 degrees C. Sucrose was only effective at -20 degrees C. It seems that these compounds act by restricting molecular diffusion. The effect of each cryostabilizer on formaldehyde binding was closely regulated by its effect on production. This is discussed in terms of the binding equation parameters. The binding of formaldehyde during frozen storage was dependent on protein rearrangements leading to reactive groups becoming available. The constraints of cryostabilizers on molecular diffusion reduced the exposure of these groups. Consequently, the interpretation of formaldehyde reactivity was biased, leading to conclusions different from those that would be obtained from a study done under standard conditions.