Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidium monoazide and real-time PCR

The increase of foodborne outbreaks associated with fresh vegetables has highlighted the importance of developing rapid and specific methods for the detection and quantification of foodborne pathogens. In this sense, real-time PCR (qPCR) fulfills these requirements although it may detect dead cells. Recently, a potential strategy to specifically detect viable cells has been proposed relying on the use of DNA binding molecules as sample pretreatment previous to the qPCR. In this study propidium monoazide (PMA) and reagent D, combined with qPCR, were evaluated for the detection and quantification of viable Escherichia coli O157:H7, Salmonella and Listeria monocytogenes. Initially, the optimal concentration of both reagents was determined for discrimination between viable and dead bacteria in cell suspensions. Although both reagents showed similar reductions for the three pathogens, reagent D was toxic to L. monocytogenes and Salmonella and therefore only PMA was used to evaluate the applicability of this technique on food samples. A final concentration of 50 μM PMA was assayed in artificially inoculated spinach and mixed salad. PMA-qPCR signal was negative for all dead cell concentrations tested except for mixed salad inoculated with L. monocytogenes at the highest concentration. These results demonstrate that PMA-qPCR is a suitable technique for the detection and quantification of viable pathogens in fresh-cut vegetables at the levels normally found in vegetable samples.     

Inactivation of Escherichia coli O157:H7 and spoilage yeasts in germicidal UV-C-irradiated and heat-treated clear apple juice

The decimal reduction times (D) of individual and composited Escherichia coli O157:H7 or spoilage yeasts in UV-C irradiated and heated (55 °C) clear apple juices (pH 3.68, 12.5 °Brix) were determined. Spoilage yeasts (D = 6.38-11.04 min) were found to be generally more UV-C resistant than E. coli O157:H7 (D = 0.5-2.76 min), while the opposite was observed in terms of thermal resistance (E. coli D=0.9-4.43 min; yeast D = 0.03-6.10 min). All spoilage yeast proliferated in the untreated juice (25 °C) while all E. coli strains were inactivated. Except for E. coli O157:H7 in UV-C irradiated apple juice, the composited inocula of both pathogenic and spoilage test organisms were less tolerant than the identified most resistant strain or species. The results of this study may be used in identifying appropriate target organisms, as well as the modes of inoculation, in challenge studies and eventually in the establishment and validation of process lethalities for apple juices and similar commodities.     

Removal of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 biofilms on stainless steel using scallop shell powder

Biofilms on steel surfaces containing Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 continue to threaten dairy and meat processors. In this study, the ability of scallop shell powder (SSP) to remove biofilms formed by these three pathogens on stainless steel plates was examined. Whey powder solution (WPS) and bench wash water (BWW) provided by dairy and meat factories, respectively, were inoculated with L. monocytogenes, S. aureus or E. coli O157:H7 (9 log₁₀ CFU/ml). Stainless steel plates (10 cm²) were placed in the inoculated fluids and incubated at 20 °C at 48 h to form biofilms. After drying and washing in sterile water, the plates were treated with 0.0, 0.25, or 0.50% (w/v) SSP slurries for 1, 5, or 10 min and then quantitatively examined for the three pathogens. Both 0.25 and 0.50% SSP reduced L. monocytogenes on the plates by 4 log CFU/cm² with a 1 min exposure to 0.50% SSP decreasing S. aureus by 5 logs CFU/cm². After 1 min in 0.25 and 0.50% SSP, E. coli O157:H7 populations in WPS and BWW biofilms decreased 4 and 6 log CFU/cm² and 3 and 5 log CFU/cm², respectively. Increasing the concentration of SSP led to significantly increased efficacy against the tested pathogens (P < 0.05). In conclusion, this study showed that SSP slurries could significantly reduce the numbers of L. monocytogenes, S. aureus and E. coli O157:H7 in biofilms on stainless steel surfaces.     

Prediction of Escherichia coli O157:H7 adhesion and potential to form biofilm under experimental conditions

Escherichia coli O:157:H7 adhesion and potential to form biofilm on three different surfaces commonly used in the food industry was evaluated using probabilistic models; the surfaces tested were stainless steel 304 (SS304), poly(vinyl chloride) film covered with thick cloth (PVC1) and poly(vinyl chloride) film covered with thin cloth (PVC2). Using a Central Composite Rotational Design (CCRD), the effect of contact time (0 h, 7 h, 24 h, 41 h and 48 h) and temperature (12 °C, 17 °C, 28 °C, 39 °C and 44 °C) on the probability of achieving a particular adherent cell count (Log₁₀ CFU cm⁻²) was determined. By analyzing response surface plots and their corresponding contour plots and by determining quadratic equations for each surface, experimental values were shown to be significant in accordance with predicted values in all cases. The adjusted determination coefficient (R_adj²) was 90.5%, 97.2% and 98.9% for SS304, PVC1 and PVC2, respectively, and the level of significance was P ≤ 0.001. The bias factor (B_f) and accuracy factor (A_f) both approached 1.0 for the three surfaces evaluated. The model equations for predicting optimum response values were verified effectively by a validation data set for all surfaces evaluated. Therefore, an RSM provides a useful and accurate method for predicting E. coli O157:H7 adhesion and potential to form biofilm on SS304, PVC1 and PVC2 and could be considered to be a standard way to ensure food safety with respect to E. coli O157:H7 contamination through adhesion and biofilm formation.     

Fate of Listeria monocytogenes and Escherichia coli O157:H7 in the presence of natural background microbiota on conventional and organic lettuce

Foodborne illnesses due to the consumption of contaminated raw vegetables is a continuing food safety concern. The limited efficacy of chlorine products to disinfect in fresh-cut industries, has led to study other methods or strategies to improve the safety of processed fresh-cut products. It has been reported that the presence of competing microorganisms on the surfaces of fresh produce can contribute to the reduction of pathogens. The aim of this study was to evaluate the interactions between the natural background microbiota of shredded conventional and organic lettuce and Listeria monocytogenes and Escherichia coli O157:H7. The effect of different initial load of background microbiota (‘low’, ‘medium’ and ‘high’) was tested for its ability to reduce L. monocytogenes and E. coli O157:H7 populations on shredded lettuce during storage at 10 ± 1 °C for 8 days. After the different pre-conditioning steps in order to obtain different initial loads of microbiota, in general, we observed that varying its size had no effect on L. monocytogenes and E. coli O157:H7 survival/growth during the storage period. Only differences on the survival/growth of L. monocytogenes and E. coli O157:H7 inoculated onto organic and conventional lettuce, respectively at the end of storage period at 10 °C were found. These results highlight the necessity for corrective measures to avoid contamination of fresh-cut vegetables with foodborne pathogens.     

Microbiological quality and safety of minimally processed fruits in the marketplace of southern Portugal

The availability of fresh-cut fruit (FCF) in the marketplace has been increasing in Portugal, although reports of its microbial quality are not known. Due to the growing concerns of these commodities over their microbial safety, the objectives of this work were to study the microbiological quality and prevalence of Salmonella and Listeria monocytogenes on fresh-cut fruits sold in southern Portugal. A study to examine the changes in pH and microbial counts, before and after the expiration dates, was also made. A total of 160 samples was purchased in the local grocery stores between September 2011 and August 2014, before their sell-by date. These samples were assayed for aerobic mesophilic (AM) and psychrotrophic (AP) microorganisms, yeasts and molds (YM), lactic-acid bacteria (LAB), coliforms (TC), Escherichia coli and coagulase positive staphylococci as well as L. monocytogenes and Salmonella. The microbiological counts ranged from 3.0-9.2 lg cfu/g (AM); 2.2–10.7 lg cfu/g (AP); 2.3–10.4 lg cfu/g (YM); 1.9–9.0 lg cfu/g (LAB) and less than 1–9.1 lg cfu/g (TC). The melons and watermelon presented the highest levels of the microbial quality parameters studied. However, no E. coli, staphylococci, Salmonella and L. monocytogenes were detected in any of the samples. After the sell-by date, an increase of the AM, AP, LAB and YM values was observed in all fruits. Conversely, the differences found in TC counts before and after the best-before date had no statistical significance. A decrease in pH was observed in all fruits except pineapple whose pH slightly increased after 14 days of storage. The results highlight the importance of preventing contamination and cross contamination, selecting adequate decontamination technologies and maintaining a strict temperature control during processing, distribution and selling of FCF.     

Comparison of intense pulsed light- and ultraviolet (UVC)-induced cell damage in Listeria monocytogenes and Escherichia coli O157:H7

The purpose of this study was to compare the degree of microbial inactivation and cell damage induced by intense pulsed light (IPL) and short-wavelength ultraviolet (UVC) in Listeria monocytogenes and Escherichia coli O157:H7. The viability of the food-borne pathogens treated with IPL and UVC (254 nm) decreased exponentially with treatment time. Particularly dramatic reductions in L. monocytogenes and E. coli O157:H7 were observed for IPL treatments at energy densities of 376 and 455 W/m², with an approximately 7-log reduction for a treatment time of 60-180 s. Also, a 4-log reduction of L. monocytogenes and a 5-log reduction of E. coli O157:H7 were achieved with UVC irradiation for 1200 s. The types and amounts of IPL- and UVC-induced DNA damage in both microorganisms were determined and compared. DNAs from cells irradiated with either IPL or UVC accumulated double-strand breaks (DSBs), single-strand breaks, and cyclobutane pyrimidine dimers, and with a similar pattern; however, more DSBs were detected following UVC than following IPL in both types of microorganism. Transmission electron microscopy observations of IPL- and UVC-induced cell damage clearly indicate that bacterial cell structures were destroyed by IPL treatment but not by UVC treatment.     

Mechanism of the combined anti-bacterial effect of green tea extract and NaCl against Staphylococcus aureus and Escherichia coli O157:H7

The mechanism of the combined anti-bacterial effect of green tea extract (GTE) and NaCl against Staphylococcus aureus NBRC 13276 and Escherichia coli O157:H7 was investigated. After treatment for 1 h, GTE was more effective against S. aureus than E. coli O157:H7, and combined GTE/NaCl treatment caused greater cellular damage in S. aureus NBRC 13276, where it was bactericidal, than E. coli O157:H7. Compared to treatment with 1.0 mg/mL GTE, which had no effect on the survival rate of E. coli O157:H7 after 48 h, treatment with 4% NaCl alone caused greater cellular damage. Moreover, bacteria pretreated with NaCl showed delayed growth in the presence of GTE. It is therefore likely that susceptibility of E. coli O157:H7 to GTE was increased by exposure to NaCl. E. coli O157:H7 pretreated with GTE and NaCl did not multiply in the presence of GTE. Visualization of the catechin components of GTE-treated bacteria using an electron microscope and SDS-PAGE analysis of cell proteins showed that GTE attached to proteins on the surface of the bacteria to form high-molecular weight complexes, suggesting the possibility that GTE inhibits the uptake and secretion of substrates and inhibits enzyme activity. Notably, after the GTE treatment for 1 h, both bacterial strains suffered injury but recovered by cultivation in rich medium. The damage and aggregation of proteins caused by GTE treatment were repaired upon treatment with LP diluent.     

The effect of different processing parameters on the efficacy of commercial post-harvest washing of minimally processed spinach and shredded lettuce

The effect of different processing parameters on the efficacy of commercial post-harvest biocidal washes to decrease the bacterial loading on spinach and lettuce has been evaluated. Sampling was performed at two spinach processors (Facility A & B) and a shredded lettuce producer (Facility C). Aerobic colony counts (ACC) and coliform counts were determined on samples taken at pre- and post-wash. In parallel, the heterotrophic plate count (HPC) and coliform levels in wash water was also determined. Processing parameters measured were the temperature of leafy greens (pre- and post-washing) and wash water. The sanitizer levels (peroxyacetic acid, oxidation-reduction potential), pH, conductivity and turbidity were also measured. The wash process in Facility B had a residence time of 50 s for the spinach, maintained a constant hypochlorite concentration and continuously re-charged the tanks with fresh water. In contrast, Facility A had a short residence time (15 s) did not maintain a constant sanitizer (peroxyacetic acid) concentration or re-charge tanks with fresh water. Despite the differences in processing operations there was no statistical difference between the log count reductions (LCR) obtained in ACC and coliform counts although counts were only reduced by <0.6 log cfu/g. The carriage of Escherichia coli on pre-wash spinach was 19% and 25% in Facility A and B respectively. There was a high prevalence (57% positive) of E. coli in the wash water of Facility A with none being recovered in water samples taken from Facility B. Yet, the carriage of E. coli on post-wash spinach was the same in the two facilities (7%). Lettuce harboured a lower level of both ACC and coliforms with LCR being significantly greater than spinach. In general, the LCR in ACC and coliforms could be positively correlated to bacterial counts of pre-washed leafy greens and conductivity (solids content) of the wash water. A negative correlation was found between LCR and water temperature. Interestingly, within the ranges measured the LCR was independent of the bacterial loading of the water. The results of the study confirmed the limited efficacy of biocidal washes to remove field acquired contamination. Although it is thought maintaining a low microbial loading in the wash water and maintaining sanitizer concentration is key the current study suggests high conductivity and low temperature of the wash water enhances the LCR achieved.     

Isolation, identification and monitoring of contaminant bacteria in Iranian Kefir type drink by 16S rDNA sequencing

Iranian Kefir type drink (IKTD) is a highly consumed, traditional Iranian, fermented milk product. To improve monitoring procedures for food safety 32 industrial Kefir type drinks from 4 brands and 8 different production dates as well as 32 samples from pasteurized milk of the same Kefir manufacturers and air of the production sites were analyzed for contaminations. 16S rDNA extraction from Kefir samples as well as 16S rDNA obtained from samples incubated on Columbia agar were analyzed using PCR/DGGE, cloning, sequencing and phylogenetic classification. Already DGGE analysis indicated contaminations including Bacillus strains. Subsequently analysis of cultured clones indicated contaminations with Bacillus sp. including Bacillus cereus, Bacillus thuringiensis and Paenibacillus sp. in 9 (28%) from all analyzed samples. Also 38% of pasteurized milk samples were contaminated with B. cereus. The average count of B. cereus was 74 ± 19 cfu/ml. B. cereus and B. thuringiensis were found as contaminant bacteria in the air of the all manufacturing sites. These results suggest that milk is one of the most important sources of contamination with Bacillus sp., especially B. cereus for Kefir products in Iran. But bacterial contamination in Kefir samples might also originate from the air of the production sites. 16S rDNA analysis accelerates monitoring strategies.     

Effectiveness of combined Pulsed Electric Field (PEF) and Manothermosonication (MTS) for the control of Listeria innocua in a smoothie type beverage

The combination of novel, non-thermal technologies for preservation purposes is a recent trend in food processing research. The objectives of the current study were (i) to optimise PEF or MTS treatment conditions which would achieve a maximum reduction of up to 3 log cycles of Listeria innocua in a milk based smoothie, when these technologies were applied individually, and (ii) to investigate possible additive or synergistic effects of the combined technologies. Microbiological analysis was performed by inoculating the smoothie with L. innocua and enumerating populations pre- and post-processing. All technologies applied within combinations significantly reduced L. innocua in the smoothie, when compared to untreated controls (p ≤ 0.0001). The sequence in which the MTS and PEF were applied was found to have a significant impact on the level of microbial reduction achieved (p ≤ 0.05). The sequence of MTS followed by PEF was the most effective in inactivating L. innocua achieving a mean reduction of 5.6 log cfu/ml, thereby exceeding the 5 log cycles minimum requirement specified by the United States Food and Drug Administration (US FDA). Significantly (p ≤ 0.05) lower reductions of 4.2 log cfu/ml were achieved when the PEF + MTS sequence combination was applied. The combination of MTS + PEF achieved inactivation comparable to thermally treated samples (p > 0.05). This study has shown the combination MTS + PEF is a promising hurdle preservation approach to control undesirable microorganisms in milk based smoothie beverages.     

Effect of Hibiscus sabdariffa extract and Nigella sativa oil on the growth and aflatoxin B1 production of Aspergillus flavus and Aspergillus parasiticus strains

This study was undertaken to evaluate the inhibitory effect of Hibiscus sabdariffa calyx extract at concentrations of 5, 7.5, 10 and 12.5 g/100 ml and Nigella sativa oil at concentrations of 1, 2 and 3 ml/100 ml on the growth and aflatoxin B₁ production by Aspergillus parasiticus (CBS 921.7) and Aspergillus flavus (SQU 21) strains. The inhibition of aflatoxin B₁ production by the different concentrations of H. sabdariffa calyx ranged between 91.5-97.9% and 87.1-93.3% for A. flavus and A. parasiticus strains, respectively, whereas the inhibition by different concentrations of N. sativa oil ranged between 47.9 and 58.3% for A. flavus and 32-48% for A. parasiticus strains. The different concentrations of H. sabdariffa calyx and N. sativa oil had no significant effect on the growth of either Aspergillus species. Neither H. sabdariffa calyx nor N. sativa oil detoxified pure aqueous aflatoxin. Our results suggest that H. sabdariffa calyx and N. sativa oil extracted from seeds had metabolic effects on aflatoxin biosynthesis pathway of both Aspergillus species and can be used as an effective biocontrol and non-toxic biopreservatives in food industry against aflatoxin contamination.     

多孔介质中CO2水合物饱和度与阻抗关系模拟实验研究

确定沉积物中天然气水合物饱和度是评估水合物资源、开发利用天然气水合物工作中的一项基础而关键的工作。利用青岛海洋地质研究所自主设计、研制的天然气水合物阻抗监测模拟实验装置,研究了CO ₂气体与去离子水在多孔介质中天然气水合物的生成与分解过程。指出：天然气水合物生成过程会使多孔介质阻抗增大;天然气水合物分解过程导致多孔介质阻抗减小。并且多孔介质的阻抗变化与反应体系的温度压力变化相互对应,能够体现天然气水合物生成与分解各阶段的特点。此外,还利用Archie公式,使用天然气水合物的阻抗值计算多孔介质中水合物的饱和度,得到了多孔介质中天然气水合物饱和度随反应时间的增长曲线。