Molecular characterization of piezotolerant and stress-resistant mutants of Staphylococcus aureus.

AIMS: In the previous work, following a pressure treatment with wild-type Staphylococcus aureus, we obtained piezotolerant isolates showing altered phenotypic characteristics. This work focuses on understanding the genetic background of their altered phenotype. METHODS AND RESULTS: AK23, a representative piezotolerant isolate was subjected to DNA microarrays, corroborated by PCR product sequencing and revealed 10-gene deletion. All other piezotolerant isolates possessed the mutation encompassing the region from SAR0665 to SAR0674 genes (9351 bp) which was most likely the result of recombination between two homologous loci (ATTGCGGGTG) present in both genes. RNA microarray transcriptomic analysis showed that due to partial deletion of the low-affinity phosphate transporter pitA, the high-affinity PhoU-PstABCS operon was upregulated in AK23 which could be the reason for piezotolerance. Furthermore, AK23 showed low levels of the virulence gene regulator rnaIII resulting in the downregulation of several agr system genes explaining the impaired virulence characteristics of the mutant. CONCLUSIONS: Naturally occurring mutations can result in piezotolerance which can be of a concern for high hydrostatic pressure-treated foods. SIGNIFICANCE AND IMPACT OF THE STUDY: A locus has been identified in piezotolerant S. aureus mutants providing insight into possible mechanisms associated with phenotypic characteristics of S. aureus. Further work should study each individual gene of the locus.

Use of Fourier transform infrared spectroscopy for monitoring the shelf life of ham slices packed with probiotic supplemented edible films after treatment with high pressure processing.

The aim of the present study was to investigate the potential use of Fourier transform infrared (FTIR) spectroscopy to quantify biochemical changes occurring in ham slices packed with probiotic supplemented edible films and treated with High Pressure Processing (HPP), in monitoring spoilage. Details regarding the data collection and experimental procedure were presented by Pavli et al. (2017). A series of Partial Least Squares (PLS) models were developed to correlate spectral data from FTIR analysis with ham spoilage during storage under vacuum at different temperatures (4, 8 and 12°C). FTIR spectra were collected from the surface of the ham samples in parallel with microbiological analysis of total viable counts (TVC) and lactic acid bacteria (LAB). Qualitative interpretation of spectral data was based on a sensory evaluation, using a hedonic scale, classifying the samples in three quality classes, fresh, semi-fresh and spoiled. The scope of the modeling approach was to discriminate the ham slices in their respective quality class and additionally to predict the microbial population directly from spectral data. The results obtained demonstrated that the processing of the samples affected the performance of classification in the sensory classes, with better results observed in the case of for ham slices packed with probiotic supplemented (PS) edible films and of control samples without HPP. The performance of PLS regression models on providing quantitative estimations of microbial counts were based on specific figures of merit (bias factor, accuracy factor, root mean square error, percentage of prediction error). Bias and accuracy factors were close to unity for both microbial groups tested for samples without HPP, whereas for HPP treated samples the values of these indices ranged from 0.963 to 1.332, depending on the case and indice. The results of this study demonstrated for the first time that although FTIR can be used reliably for the rapid assessment of sliced ham, additional processes such as HPP can affect its performance.

Effect of temperature and water activity on growth and ochratoxin A production boundaries of two Aspergillus carbonarius isolates on a simulated grape juice medium.

AIMS: To develop and validate a logistic regression model to predict the growth and ochratoxin A (OTA) production boundaries of two Aspergillus carbonarius isolates on a synthetic grape juice medium as a function of temperature and water activity (a(w)). METHODS AND RESULTS: A full factorial design was followed between the factors considered. The a(w) levels assayed were 0.850, 0.880, 0.900, 0.920, 0.940, 0.960, 0.980 and the incubation temperatures were 10, 15, 20, 25, 30, 35 and 40 degrees C. Growth and OTA production responses were evaluated for a period of 25 days. Regarding growth boundaries, the degree of agreement between predictions and observations was >99% concordant for both isolates. The erroneously predicted growth cases were 3.4-4.1% false-positives and 0.7-1.4% false-negatives. No growth was observed at 10 degrees C and 40 degrees C for all a(w) levels assayed, with the exception of 0.980 a(w)/40 degrees C, where weak growth was observed. Similarly, OTA production was correctly predicted with a concordance rate >98% for the two isolates with 0.7-1.4% accounting for false-positives and 2.0-2.7% false-negatives. No OTA production was detected at 10 degrees C or 40 degrees C regardless of a(w), and at 0.850 a(w) at all incubation temperatures. With respect to time, the OTA production boundary shifted to lower temperatures (15-20 degrees C) as opposed to the growth boundary that shifted to higher temperature levels (25-30 degrees C). Using two literature datasets for growth and OTA production of A. carbonarius on the same growth medium, the logistic model gave one false-positive and three false-negative predictions out of 68 growth cases and 13 false-positive predictions out of 45 OTA production cases. CONCLUSIONS: The results of this study suggest that the logistic regression model can be successfully used to predict growth and OTA production interfaces for A. carbonarius in relation to temperature and a(w). SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed modelling approach helps the understanding of fungal-food ecosystem relations and it could be employed in risk analysis implementation plans to predict the risk of contamination of grapes and grape products by A. carbonarius.

Temperature-assisted high hydrostatic pressure inactivation of Staphylococcus aureus in a ham model system: evaluation in selective and nonselective medium.

AIMS: The purpose of this study was to investigate the inactivation kinetics of Staphylococcus aureus in a ham model system by high hydrostatic pressure at ambient (25 degrees C) and selected temperatures (45, 55 degrees C). Selective [Baird Parker (BP) agar] and nonselective [brain heart infusion (BHI) agar] growth media were used for enumeration in order to count viable and sublethally injured cells. METHODS AND RESULTS: The micro-organism was exposed to a range of pressures (450, 500, 550, 600 MPa) at ambient temperature (25 degrees C) for up to 45 min. Additionally, the behaviour of the micro-organism was evaluated at mild temperatures in combination with high pressure treatment, namely: (i) 350, 400 and 450 MPa at 45 degrees C; and (ii) 350 and 400 MPa at 55 degrees C, for up to 12 min. Inactivation kinetics were calculated in terms of D(p) and z(p) values. Survival curves of S. aureus at ambient temperature were mostly linear, whereas when temperature was applied, tailing was observed in most survival curves. The estimated D(p) values and therefore the number of surviving cells, were substantially higher on the selective BP agar in the whole range of pressures applied, indicating that S. aureus showed greater recovery in the selective BP agar than the nonselective BHI agar. Samples pressurized at ambient temperature needed higher pressures (over 500 MPa) to achieve a reduction of the population of the pathogen more than 5 log CFU ml(-1). The same level of inactivation was achieved at lower pressure levels when mild heating was simultaneously applied. Indeed, more than 6 log CFU ml(-1) reductions were obtained at 400 MPa and 55 degrees C within the first 7 min of the process in BHI medium. CONCLUSION: Elevated temperatures allowed lower pressure levels and shorter processing times of pathogen inactivation than at room temperature. Greater recovery of the pathogen was observed in the selective (BP agar) medium, regardless of pressure and temperature applied. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained kinetics could be employed by the industry in selecting optimum pressure/temperature processing conditions. Attention must be given to the selection of the enumeration medium, as the use of an inappropriate medium would lead to underestimation of the surviving cells, thus imposing a risk in the microbiological safety of the product.

Modelling the effect of temperature and water activity on the growth of two ochratoxigenic strains of Aspergillus carbonarius from Greek wine grapes.

AIMS: To develop descriptive models for the combined effect of temperature (10-40 degrees C) and water activity (0.850-0.980) on the growth of two ochratoxin A producing strains of Aspergillus carbonarius from Greek wine grapes on a synthetic grape juice medium. METHODS AND RESULTS: Fungal growth was measured as changes in colony diameter on a daily basis. The maximum specific colony growth rates (mu(max)) were determined by fitting the primary model of Baranyi describing the change in colony diameter (mm) with respect to time (days). Secondary models, relating mu(max) with temperature and a(w) were developed and comparatively evaluated based on polynomial, Parra, Miles, Davey and Rosso equations. No growth was observed at 0.850 a(w) (water activity) regardless of temperature, as well as at marginal temperature levels assayed (10 and 40 degrees C) regardless of water activity. The data set was fitted successfully in all models as indicated by the values of regression coefficients and root mean square error. Models with biological interpretable parameters were highly rated compared with the polynomial model, providing realistic cardinal values for temperature and a(w). The optimum values for growth were found in the range 0.960-0.970 a(w) and 34-35 degrees C respectively for both strains. The developed models were validated on independently derived data from the literature and presented reasonably good predictions as inferred by graphical plots and statistical indices (bias and accuracy factors). CONCLUSIONS: The effect of temperature and a(w) on the growth of A. carbonarius strains could be satisfactorily predicted under the current experimental conditions, and the proposed models could serve as a tool for this purpose. SIGNIFICANCE AND IMPACT OF THE STUDY: The results could be successfully employed as an empirical approach in the development and prediction of risk models of contamination of grapes and grape products by A. carbonarius.

Modelling the effect of high pressure on the inactivation kinetics of a pressure-resistant strain of Pediococcus damnosus in phosphate buffer and gilt-head seabream (Sparus aurata).

AIMS: The aim of this research was to: (i) determine the inactivation pattern of a pressure-resistant strain of Pediococcus damnosus by high hydrostatic pressure in phosphate buffer (pH 6.7) and gilt-head seabream using the linear, biphasic and Weibull models; and (ii) validate the applicability of the Weibull model to predict survival curves at other experimental pressure levels. METHODS AND RESULTS: A pressure-resistant strain of P. damnosus was exposed to a range of pressures (500, 550, 600 and 650 MPa) in phosphate buffer (pH 6.7) and gilt-head seabream for up to 8 min at ambient temperature (23 degrees C). Inactivation kinetics were described by the linear, biphasic and Weibull models. Increasing the magnitude of the pressure applied resulted in increasing levels of inactivation. Pronounced tailing effect was observed at pressures over 600 MPa. The Weibull and biphasic models consistently produced better fit than the linear model as inferred by the values of the root mean squared error, coefficient of determination (R2) and accuracy factor (A(f)). The scale factor (b) of the Weibull model was linearly correlated with pressure (P) treatment in the whole pressure range. Substituting the b parameter in the initial Weibull function and calculating the shape factor (n) by linear interpolation, high pressure (P) was directly incorporated into the model providing reasonable predictions of the survival curves at 570 and 630 MPa. Comparison between the survival curves in phosphate buffer and gilt-head seabream showed a clear protective effect of the food matrix on the resistance of the micro-organism, especially at 500 and 550 MPa. CONCLUSIONS: The Weibull and biphasic models were more flexible to describe the survival curves of P. damnosus in the experimental pressure range, taking also into account the tailing effect that could not be included in the linear model. The Weibull model could also give reasonable predictions of the survival curves at other experimental pressures in both pressure menstrua. As the food matrix has a protective effect in microbial inactivation, the development of accurate mathematical models should be done directly on real food to avoid under- or over-processing times. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of accurate models to describe the survival curves of micro-organisms under high hydrostatic pressure treatment would be very important to the food industry for process optimisation, food safety and extension of the applicability of high pressure processing.

Physicochemical, microbiological, and organoleptic profiles of Greek table olives from retail outlets.

The physicochemical, microbiological, and organoleptic profile of different commercial table olive products from retail outlets was studied. Average pH values were 4.00, 3.96, and 4.31 for Spanish-style green, naturally black, and dry-salted olives, respectively, while salt content was 6.21, 7.34, and 8.00% for the same commercial products. Mean values for titratable acidity were 0.53 and 0.63% (wt/vol) for green and naturally black olives. In general, mean values for pH, titratable acidity, and salt content were in accordance with the requirements established by the International Olive Oil Council (IOOC) for the trade of table olives, although considerable variation was observed within individual olive samples. Salt content of dry-salted olives did not meet the minimum limit of 10% established by the IOOC. The dominant microbiota consisted of lactic acid bacteria and yeasts. Their population was less than 10(9) CFU ml(-1), as stipulated by the IOOC standard for fermented olives held in bulk in a covering liquid. These microorganisms come from the natural microbiota found in spontaneous fermentations and impose no risk to human health. No enterobacteria, pseudomonads, Bacillus cereus, or Clostridium perfringens were detected in any of the samples given the physicochemical characteristics found. The organoleptic profile varied greatly according to processing style and commercial preparation. Green olives had more uniform sensory characteristics than naturally black and dry-salted olives. The most important attributes that influenced the judgment of the panelists were salt content and crispness of the olives.

Effect of prestorage treatmlents and storage conditions on the survival of Salmonella enteritidis PT4 and Listeria monocytogenes on fresh marine and freshwater aquaaculture fish.

The effect of prestorage treatments, such as immersion in a sorbate solution (5%, wt/vol), heating (60 degrees C, 1 min), and a combination of the two treatments, and the subsequent storage in air or under modified atmosphere packaging (MAP; 40% CO2, 30% O2, and 30% N2) at chill temperatures (0 +/- 1 degrees C), on Listeria monocytogenes and Salmonella Enteritidis PT4 was studied. The prestorage treatments affected the pathogenic bacteria, and in all cases, there was a decrease in their population, with the sorbate and combination (hot water and sorbate) treatment being most effective. The beneficial effect of the prestorage treatments, which was more pronounced in storage under MAP conditions, suggests an interaction of the treatments with the CO2 of MAP against injured bacterial cells.

Survival of Staphylococcus aureus and Salmonella enteritidis on salted sardines (Sardina pilchardus) during ripening.

The ripening period for salted sardines ranges from 4 to 6 months, depending on the season. Sometimes producing industries need to distribute the product earlier owing to market needs, and when this happens the product's safety needs to be assured. The purpose of this work was to study the survival of Staphylococcus aureus and Salmonella Enteritidis on salted sardines during a ripening period of 115 days. Salted sardines were inoculated with pure cultures of S. aureus and Salmonella Enteritidis (10(5) CFU/g of fish on day 0). After 5 days of ripening, the water activity value for the sardines decreased from 0.93 to 0.69. The survival of both pathogens and that of total viable cells were evaluated during the ripening process. Total viable counts decreased by 2 log units over the 115-day ripening period. Salmonella Enteritidis and S. aureus survived for 60 and 90 days, respectively. Therefore, the use of a 90-day ripening period could be effective in assuring the safety of the final product.

Microbial colonization of naturally black olives during fermentation and associated biochemical activities in the cover brine.

AIMS: To establish the site of microbial growth on naturally black fermented table olives, and to monitor the population dynamics of yeasts and selected micro-organisms together with the changes in organic acid profile and pH in the cover brine during fermentation. METHODS AND RESULTS: During fermentation, the numbers of Enterobacteriaceae and Pseudomonas spp. in the brine decreased whilst lactic acid bacteria and yeast populations increased. Scanning electron microscopy showed that a yeast-rich biofilm developed on the epicuticular wax of the olive skin during fermentation. Yeasts also predominated in the stomatal openings, but bacteria were more numerous in intercellular spaces in the sub-stomatal flesh. Citric, malic and tartaric acids were the major organic acids accumulating in the brine during fermentation. CONCLUSIONS: Micro-organisms associated with the skin, stomata and flesh in fermenting black olives may experience different local conditions to those prevailing in the cover brine. SIGNIFICANCE AND IMPACT OF THE STUDY: These are the first observations of the micro-organisms associated with the fruit of naturally fermented black olives and of the accumulation of specific organic acids during fermentation.

Modelling the effectiveness of a natural antimicrobial on Salmonella enteritidis as a function of concentration, temperature and pH, using conductance measurements.

The growth of Salmonella enteritidis in a brain heart infusion medium was monitored using the traditional viable count method and by conductance measurements using a Rabit impedance instrument. Growth curves (log10 cfu ml-1 vs time) at three different concentrations of oleuropein (0, 0.2 and 0.8%), pH values in the range of 5-8 and incubation temperatures from 22 to 42 degrees C were modelled using the Gompertz equation. A good correlation between the maximum growth rate from the viable count method and the maximum slope of the conductance curve from the impedance instrument was established. Based on this correlation, the maximum specific growth rate of Salm. enteritidis was modelled as a function of the oleuropein concentration, initial pH values and the incubation temperature with a quadratic equation, using a new, large dataset of growth measurements by conductance. The developed model was validated by statistical comparison of predicted growth rates with growth rates determined by the viable count method, within the limits of the antimicrobial, pH and temperature domain.

Growth/survival of Salmonella enteritidis on fresh poultry and fish stored under vacuum or modified atmosphere.

The effect of vacuum and modified atmosphere packaging on the growth/survival of Salmonella enteritidis on fresh poultry and fish (Boops boops) is described. Salmonella enteritidis survived but did not grow significantly in all samples (poultry or fish) at 3 degrees C. At 10 degrees C the numbers of Salm. enteritidis increases rapidly in vacuum-packed samples and in samples flushed with 100% N2, 20% CO2/80% O2 of both types of proteinaceous food. Growth was also evident in fish and poultry flushed with 100% CO2; however the rate of growth was greater in fish samples rather than in poultry.

Inhibition of Resident Microbial Flora and Pathogen Inocula on Cold Fresh Fish Fillets in Olive Oil, Oregano, and Lemon Juice under Modified Atmosphere or Air.

The development of a microbial population was studied in Mediterranean gilt-head seabream ( Sparus aurata , tsipoura in Hellenic) dressed with olive oil, lemon juice, and oregano, inoculated with Staphylococcus aureus and Salmonella enteritidis , and stored under a modified atmosphere (MA) of 40% CO2, 30% O2, and 30% N2 or air at 0 ± 1°C. The treatment had bacteriostatic and bactericidal effects on both inoculated pathogens as well as on the autochthonous flora. Brochothrix thermosphacta and pseudomonads dominated the spoilage flora under MA and under air respectively. Shewanella putrefaciens was clearly inhibited.

Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4 degrees and 10 degrees C.

The effect of mint (Mentha piperita) essential oil (0.5, 1.0, 1.5 and 2.0%, v/w) on Salmonella enteritidis and Listeria monocytogenes in a culture medium and three model foods; tzatziki (pH 4.5), taramosalata (pH 5.0) and pâté (pH 6.8), inoculated at 10(7) cfu g-1, at 4 degrees and 10 degrees C for ca 1 week was studied. In the culture medium supplemented with the essential oil, no growth was observed over 2 d at 30 degrees C determined by a conductance method with a Malthus 2000 growth analyser. Salmonella enteritidis died in tzatziki in all treatments and declined in the other foods except for pâté at 10 degrees C as judged with viable counts. Listeria monocytogenes populations showed a declining trend towards the end of the storage period but was increased in pâté. Mint essential oil antibacterial action depended mainly on its concentration, food pH, composition, storage temperature and the nature of the micro-organism.

Inhibition of Salmonella enteritidis by oleuropein in broth and in a model food system.

The inhibitory effect of commercial 'pure' oleuropein was tested against Salmonella enteritidis in a coliform broth and in reconstituted milk (model food system). It was found that the inhibition of this organism in the broth was influenced by the initial inoculum size, the pH of the medium and the concentration of additive. The inhibition was more pronounced in samples with low pH and low inoculum size. No such inhibition was evident in the model food system.

Inhibition of Staphylococcus aureus by Olive Phenolics in Broth and in a Model Food System.

The commercial 'pure' oleuropein and phenolics extracted from olives inhibited the growth and enterotoxin production by Staphylococcus aureus S-6 in broth as well as in reconstituted milk (model food system). It was found that the inhibition of this organism in N-Z amine A broth was influenced by the initial inoculum size, the pH of the media, and the concentration of additive. In particular, growth and enterotoxin B production by S. aureus were inhibited in broth with a high concentration of oleuropein (0.6%). The inhibition was more pronounced in samples with low pH and low inoculum size. In the case of milk, enterotoxin B production was also influenced by the initial concentration of extract.

Effect of phenolic compounds and oleuropein on the germination of Bacillus cereus T spores.

The phenolic compounds extracted from olives with ethyl acetate inhibited germination and outgrowth of Bacillus cereus T spores. Purified oleuropein, a well-characterized component of olive extract, inhibited these processes also. The addition of oleuropein and olive extracts 3 or 5 min after germination began, immediately decreased the rate of change of phase bright to phase dark spores and delayed significantly outgrowth.