Food Hygiene and Food Safety

In the current study, we demonstrate that E. coli O104:H4 strain C227/11Φcu, a derivative of the 2011 enterohemorrhagic/enteroaggregative (EHEC/EAEC) E. coli outbreak strain, migrated into the edible portion of lamb’s lettuce plants upon contamination of the surrounding soil. Seeds were surface-sterilized and cultivated on Murashige-Skoog agar or in autoclaved agricultural soil. Migration into the edible portions was investigated by inoculating the agar or soil close to the plants with 10⁸ colony-forming units (CFU). The edible parts, which did not come into contact with the contaminated medium or soil, were quantitatively analyzed for the presence of bacteria after 2, 4 and 8 weeks. Strain C227/11Φcu could colonize lamb’s lettuce when contamination of medium or soil occurs. The highest recovery rate (27%) was found for lettuce cultivated in agar, and up to 1.6 × 10³ CFU/g lettuce was detected. The recovery rate was lower for the soil samples (9% and 13.5%). Although the used contamination levels were high, migration of C227/11Φcu from the soil into the edible parts was demonstrated. This study further highlights the risk of crop plant contamination with pathogenic E. coli upon soil contamination. Full article

Bacterial lipopolysaccharide (LPS) in the aquatic environment has been reported to cause diseases in red swamp crayfish (Procambarus clarkii). In addition, deoxynivalenol (DON) is one of the primary mycotoxins found in aquaculture. However, the potential synergistic toxic effects of LPS and DON on crayfish are yet to be fully elucidated. In this study, crayfish were exposed to LPS (1 mg kg⁻¹), DON (3 mg kg⁻¹), and their combination (1 mg kg⁻¹ LPS + 3 mg kg⁻¹ DON, L+D) for a duration of six days. Co-exposure to LPS and DON exhibited the lowest survival rate compared to the control or individual treatments with LPS or DON alone. In the initial stage of the experiment, the combined treatment of LPS and DON showed a more pronounced up-regulation of antioxidant and immune-related enzymes in the sera compared to the other treatment groups, with a fold change ranging from 1.3 to 15. In addition, the (L+D) treatment group showed a down-regulation of immune-related genes, as well as Toll pathway-related genes in the hepatopancreas compared to LPS or DON. Moreover, the (L+D) treatment group demonstrated a 100% incidence of histopathological changes in the hepatopancreas, which were significantly more severe compared to the other three groups. In conclusion, our study provides physiological and histopathological evidence that the co-exposure to LPS and DON exerted synergistic toxic effects on crayfish. The observed effects could potentially hinder the development of the crayfish aquaculture industry in China. Full article

Aflatoxins and fumonisins, commonly found in maize and maize-derived products, frequently co-occur and can cause dangerous illness in humans and animals if ingested in large amounts. Efforts are being made to develop suitable analytical methods for screening that can rapidly detect mycotoxins in order to prevent illness through early detection. A method for classifying contaminated maize by applying hyperspectral imaging techniques including reflectance in the visible and near-infrared (VNIR) and short-wave infrared (SWIR) regions, and fluorescence was investigated. Machine learning classification models in combination with different preprocessing methods were applied to screen ground maize samples for naturally occurring aflatoxin and fumonisin as single contaminants and as co-contaminants. Partial least squares–discriminant analysis (PLS-DA) and support vector machine (SVM) with the radial basis function (RBF) kernel were employed as classification models using cut-off values of each mycotoxin. The classification performance of the SVM was better than that of PLS-DA, and the highest classification accuracies for fluorescence, VNIR, and SWIR were 89.1%, 71.7%, and 95.7%, respectively. SWIR imaging with the SVM model resulted in higher classification accuracies compared to the fluorescence and VNIR models, suggesting that as an alternative to conventional wet chemical methods, the hyperspectral SWIR imaging detection model may be the more effective and efficient analytical tool for mycotoxin analysis compared to fluorescence or VNIR imaging models. These methods represent a food safety screening tool capable of rapidly detecting mycotoxins in maize or other food ingredients consumed by animals or humans. Full article

On 6 July 2018, the Center for Epidemiology and Public Health of the French Armed Forces was informed of an outbreak of acute gastroenteritis among customers of a dining facility at a military base in Brittany, France. A total of 200 patients were reported out of a population of 1700 (attack rate: 12%). The symptoms were mainly lower digestive tract disorders and occurred rapidly after lunch on 5 July (median incubation period: 3.3 h), suggesting a toxin-like pathogenic process. A case–control survey was carried out (92 cases and 113 controls). Statistical analysis pointed to the chili con carne served at lunch on 5 July as the very likely source of poisoning. Phytohaemagglutinin, a plant lectin, was found in the chili con carne at a concentration above the potentially toxic dose (400 HAU/gram). The raw kidney beans incorporated in the chili con carne presented a high haemagglutination activity (66,667 HAU/gram). They were undercooked, and the phytohaemagglutinin was not completely destroyed. FBDOs due to PHA are poorly documented. This study highlights the need to develop methods for routine testing of plant toxins in food matrices. Improved diagnostic capabilities would likely lead to better documentation, epidemiology, and prevention of food-borne illnesses caused by plant toxins. Full article

To evaluate the decontamination effect of Inspexx 210, a cold-water disinfectant containing peracetic acid and peroctanoic acid (0.16%), knives contaminated with E. coli ATCC 25922 and S. aureus ATCC 29213 as well as naturally contaminated knives collected from different slaughtering steps of an abattoir were examined. Treatment with Inspexx 210 for 15 s reduced mean E. coli counts by 5.95 log CFU/cm² and mean S. aureus counts by 6.24 log CFU/cm². Contamination of the knives first with fat and thereafter with the bacteria led to lower mean reductions of 5.52 log CFU/cm² for E. coli and 3.58 log CFU/cm² for S. aureus. Contamination first with blood and thereafter with the bacteria had the greatest negative impact on the inactivation effect, with mean reductions of 3.70 log CFU/cm² for E. coli and 1.55 log CFU/cm² for S. aureus. Rinsing the blood with tap water before contamination with S. aureus led to the reduction of 5.50 log CFU/cm². Treatment with Inspexx 210 was also assessed for naturally contaminated knives from an abattoir. After treatment under routine conditions, colony counts were 0.09 log CFU/cm² (pigs) and 0.35 log CFU/cm² (sheep) in the wet area and below detection limit (pigs) and 0.91 (sheep) in the clean area. This study shows that Inspexx 210 can be an adequate alternative to knife decontamination with 82 °C hot water, but pre-rinsing is recommended especially in dirty process steps with a high contamination of blood and fat. Full article

The commercialization of industrial genetically modified microorganisms (GMMs) has highlighted their impact on public health and the environment. Rapid and effective monitoring methods detecting live GMMs are essential to enhance current safety management protocols. This study aims to develop a novel cell-direct quantitative polymerase chain reaction (qPCR) method targeting two antibiotic-resistant genes, KmR and nptII, conferring resistance against kanamycin and neomycin, along with propidium monoazide, to precisely detect viable Escherichia coli. The E. coli single-copy taxon-specific gene of D-1-deoxyxylulose 5-phosphate synthase (dxs) was used as the internal control. The qPCR assays demonstrated good performance, with dual-plex primer/probe combinations exhibiting specificity, absence of matrix effects, linear dynamic ranges with acceptable amplification efficiencies, and repeatability for DNA, cells, and PMA-treated cells targeting KmR/dxs and nptII/dxs. Following the PMA-qPCR assays, the viable cell counts for KmR-resistant and nptII-resistant E. coli strains exhibited a bias% of 24.09% and 0.49%, respectively, which were within the acceptable limit of ±25%, as specified by the European Network of GMO Laboratories. This method successfully established detection limits of 69 and 67 viable genetically modified E. coli cells targeting KmR and nptII, respectively. This provides a feasible monitoring approach as an alternative to DNA processing techniques to detect viable GMMs. Full article

Ptaquiloside, a naturally occurring cancer-causing substance in bracken fern, has been detected in the meat and milk of cows fed a diet containing bracken fern. A rapid and sensitive method for the quantitative analysis of ptaquiloside in bracken fern, meat, and dairy products was developed using the QuEChERS method and liquid chromatography–tandem mass spectrometry. The method was validated according to the Association of Official Analytical Chemists guidelines and met the criteria. A single matrix-matched calibration method with bracken fern has been proposed, which is a novel strategy that uses one calibration for multiple matrices. The calibration curve ranged from 0.1 to 50 µg/kg and showed good linearity (r² > 0.99). The limits of detection and quantification were 0.03 and 0.09 µg/kg, respectively. The intraday and interday accuracies were 83.5–98.5%, and the precision was <9.0%. This method was used for the monitoring and exposure assessment of ptaquiloside in all routes of exposure. A total of 0.1 µg/kg of ptaquiloside was detected in free-range beef, and the daily dietary exposure of South Koreans to ptaquiloside was estimated at up to 3.0 × 10⁻⁵ µg/kg b.w./day. The significance of this study is to evaluate commercially available products in which ptaquiloside may be present, to monitor consumer safety. Full article

The effects of exposure to heavy metals (HMs) in rice on human health have become a global public health concern, particularly in countries where rice is consumed as a staple food. The concentrations of HMs, including cadmium (Cd), arsenic (As), lead (Pb), and copper (Cu), in commercial rice samples (n = 170) were analyzed to estimate the HM exposure of consumers in Nepal. The geometric mean concentrations of Cd, As, Pb, and Cu in commercial rice were 15.5 ± 16.0, 43.4 ± 19.6, 16.0 ± 14.0, and 1066 ± 1210 μg/kg, respectively, all below the maximum allowable concentrations (MACs) recommended by FAO/WHO. Generally, the average estimated daily intakes (EDIs) of Cd, As, Pb, and Cu were all below the oral reference doses (RfDs). However, young age groups were exposed to high levels of HMs, and the average EDI of As and the P99.9 EDIs of Cu and Cd were above the corresponding RfDs. The mean hazard index and total carcinogenic risk were 1.13 and 1.04 × 10⁻³ respectively, suggesting a potential non-carcinogenic risk (NCR) and a carcinogenic risk (CR) via rice consumption. Arsenic contributed the most strongly to NCR and Cd to CR. Overall, although the HM levels in rice were generally safe, the Nepalese population may be exposed to an elevated health risk from rice consumption. Full article

N-glycolylneuraminic acid (Neu5Gc) is a specific factor in red meat that induces intestinal disease. Our aim was to investigate the effect of Neu5Gc on the intestinal barrier as well as its mechanism of endocytosis and exocytosis. Ten specific inhibitors were used to explore the mechanism of Neu5Gc endocytosis and exocytosis by Caco-2 cells. Amiloride hydrochloride and cytochalasin D had the strongest inhibitory effect on the endocytosis of Neu5Gc. Sodium azide, dynasore, chlorpromazine hydrochloride, and nystatin also inhibited Neu5Gc endocytosis. Dynasore exhibited a stronger inhibitory effect than that of chlorpromazine hydrochloride or nystatin alone. Exocytosis inhibitors, including nocodazole, brefeldin A, monensin, and bafilomycin A, inhibited the transmembrane transport of Neu5Gc. Monensin promoted the exocytosis of Neu5Gc from Caco-2 cells. In another experiment, we observed no significant inhibitory effects of monensin and brefeldin A. Dietary concentrations of Neu5Gc induced prominent damage to intestinal tight junction proteins zonula occludens-1 (ZO-1), occludin, and claudin-1 and promoted the phosphorylation of IκB-α and P65 to activate the canonical Nuclear Factor kappa-B (NF-κB) pathway. Neu5Gc increased the RNA levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α and inhibited those of anti-inflammatory factors TGF-β and IL-10. BAY, an NF-κB signaling pathway inhibitor, attenuated these changes. Reductions in the levels of ZO-1, occludin, and claudin-1 were recovered in response to BAY. Our data reveal the endocytosis and exocytosis mechanism of Neu5Gc and prove that Neu5Gc can activate the canonical NF-κB signaling pathway, regulate the transcription of inflammatory factors, thereby damaging intestinal barrier function. Full article

The aim of this study was to investigate the level of strain variability amongst food and clinical Listeria monocytogenes isolates growing at low temperatures (4 and 7 °C) in both laboratory media and real food matrices. Isolates (n = 150) grown in laboratory media demonstrated a large variation in growth profiles measured using optical density. Overall, it was noted that clinical isolates exhibited a significantly higher growth rate (p ≤ 0.05) at 7 °C than the other isolates. Analysis of variance (ANOVA) tests of isolates grouped using Multi Locus Sequence Typing (MLST) revealed that clonal complex 18 (CC18) isolates were significantly (p ≤ 0.05) faster growing at 4 °C than other CC-type isolates while CC101, CC18, CC8, CC37 and CC14 were faster growing than other CC types at 7 °C. Euclidean distance and Ward method-based hierarchical clustering of mean growth rates classified 33.33% of isolates as faster growing. Fast and slow growing representative isolates were selected from the cluster analysis and growth rates were determined using plate count data in laboratory media and model food matrices. In agreement with the optical density experiments, CC18 isolates were faster and CC121 isolates were slower than other CC types in laboratory media, UHT milk and fish pie. The same trend was observed in chocolate milk but the differences were not statistically significant. Moreover, pan-genome analysis (Scoary) of isolate genome sequences only identified six genes of unknown function associated with increased cold tolerance while failing to identify any known cold tolerance genes. Overall, an association that was consistent in laboratory media and real food matrices was demonstrated between isolate CC type and increased cold tolerance. Full article

Background microorganism growth on Chromogenic Coliform Agar (CCA) can be challenging. For this reason, a new alternative method with a Cefsulodin/Vancomycin (CV)-supplemented CCA should be developed in this study. CCA supplemented with CV was validated according to ÖNORM EN ISO 16140-4:2021 using water from natural sources in Styria, Austria. Results show that the alternative method using the supplemented CCA has similar values in relation to sensitivity (82.2%), specificity (98.6%) and higher selectivity (59%) compared to the reference method. Repeatability and reproducibility were acceptable for the alternative method and showed similar results with the reference method. The alternative method shows a very low false positive rate and a low false negative rate paired with good performance regarding the inclusion study. The exclusion study shows the advantage of our method by suppressing background microorganisms and facilitating the process of enumeration of Escherichia coli and other coliform bacteria on CCA plates. Aeromonas hydrophila and Pseudomonas aeruginosa growth was inhibited using the supplement. To conclude, the coliform CV selective supplement combined with CCA is an appropriate tool for coliform bacteria detection in water samples. Full article