Occurrence of Alternaria toxins in food products in The Netherlands

Alternaria toxins are mycotoxins that can contaminate cereals, oilseeds and various fruits and vegetables such as apples, tomatoes, citrus fruits and olives. The fungi can grow at low temperatures, thus causing spoilage even during transport and storage. Currently, there are relatively little occurrence data on Alternaria toxins in food products and in the Netherlands most data are limited to the presence of alternariol (AOH) and alternariol methyl ether (AME) in cereals. Tenuazonic acid (TeA), tentoxin (TEN) and altenuene (ALT) have been recently identified by the Standing Committee on the food Chain and animal health as Alternaria toxins of concern. A survey (95 samples) was conducted in the Netherlands in 2013. The aim was to screen the levels of the five Alternaria toxins in wine (n = 5), fresh apples (n = 11), apple juices (n = 7), fresh tomatoes (n = 19), tomato sauces (n = 8), fresh citrus (n = 11), dried figs (n = 5), olives (n = 10), sunflower seeds (n = 5) and cereals (n = 14). Multi-mycotoxin methods for the analysis of mycotoxins were adapted for this purpose. Their performance characteristics were assessed and were as follows: recoveries and precision ranged from 85 to 112% and from 4 to 20%, respectively. LOQs were between 1.5 and 5.0 μg kg⁻¹. In the subsequent survey, AOH, AME, TeA, and TEN were detected in one or more food commodities, while ALT was not detected in any of the samples. TeA was found in 27% of the samples and at relatively high concentrations in sunflower seeds, tomato sauces and dried figs (up to 2345 μg kg⁻¹). Alternaria toxins occurred regularly in cereals, tomato sauces, figs, wine and sunflower seeds. Only incidental occurrence of the Alternaria toxins was observed in fresh apples, fresh citrus fruits, fresh tomatoes and olives.     

Mycobiota and toxicogenic Alternaria spp. strains in Malbec wine grapes from DOC San Rafael,Mendoza, Argentina

Alternaria, one of the most mycotoxigenic genus commonly found in wine grapes, could represent a high risk for wine consumer's health. The aims of this work were to identify the mycobiota of Malbec wine grapes under the influence of routine control viticulture practices, to identify Alternaria spp. strains by morphological and molecular methods and characterize their toxicogenic ability and pathogenicity. Alternaria was the main component of the wine grape mycobiota of the DOC San Rafael at harvest time (81%) followed by Cladosporium (7%) and only in minor percentage by Penicillium (4%) and Aspergillus (3%) among others. The application of an organic or non-organic treatment in the vineyard did not affect significantly the incidence of the present genera. According to morphological and molecular identification based on the genetic marker Alt a 1, all Alternaria isolates were included into Alternaria alternata species. Of 34 analyzed Alternaria strains, 97% were able to produce at least one of the three mycotoxins analyzed: alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) and 53% co-produced the three mycotoxins. TA was the toxin produced at highest frequency (97%) and at highest levels in a range from 11.2 to 1941.0 ppm. It was followed by AOH produced by 71% of the strains, in a range from 1.8 to 437.0 ppm and AME produced by 59% of the strains, in a range from 0.6 ppm to 663.4 ppm. The 55% of the Alternaria strains were very pathogenic, 31% moderately pathogenic and only 14% were slightly pathogenic. In the present work, a high incidence and prevalence of Alternaria genus was reported despite the use of routine control viticulture practices, as well as a high percentage of toxicogenic and pathogenic Alternaria strains.     

Alternaria toxins and conjugates in selected foods in the Netherlands

A survey on Alternaria toxins in the food categories dried figs (n = 14), sunflower products (n = 24) and tomato products (n = 43) was carried out in the Netherlands on samples collected in retail stores in autumn 2014. The occurrence data from this survey confirmed the previously reported data of a pilot survey in 2013, with tenuazonic acid being an ubiquitous Alternaria toxin in dried figs (100% of the samples), sunflower seeds (80% of the samples) and tomato products (60% of samples) at relatively high concentrations, up to 1728 μg/kg in dried figs and 1350 μg/kg in sunflower seeds. Despite the occurring of high concentrations of tenuazonic acid in sunflower seeds and figs, it is unlikely that the population in the Netherlands is exposed to levels of concern. Alternariol was detected in 27% of the tomato products ranging from LOQ (2 μg/kg) up to 26 μg/kg, while alternariol monomethyl ether was present in 7% of the tomato products. In the worst case situation, consumption of these products may result in exposure at levels above the threshold of toxicological concern of 2.5 ng/kg body weight/day for alternariol. None of seven conjugates of Alternaria toxins was detected above the limit of quantification (LOQ) of 2.5 μg/kg.     

Real-time PCR with internal amplification control for the detection of Cronobacter spp. (Enterobacter sakazakii) in food samples

Cronobacter spp. (Enterobacter sakazakii) is an opportunistic pathogen and is linked with life-threatening infections in neonates. The organism has been isolated from a wide variety of foods and environments. In this study, a Taqman real-time PCR assay incorporating an internal amplification control (IAC) was developed and evaluated for specific detection of Cronobacter spp. in foods. Previously reported macromolecular synthesis (MMS) operon sequence was selected for specificity, and 67 bacterial strains, including four strains of Cronobacter spp., were evaluated. All Cronobacter strains were successfully identified, however no cross-reactivity was observed with non-Cronobacter strains. Detection limit of the assay in pure culture and formula infant without enrichment was 1.2 × 10³ CFU/ml (1.2 × 10¹ CFU/assay). After 24 h enrichment in broth, as few as 10⁰ CFU/ml or g of Cronobacter could be detected in artificially contaminated food samples (infant formula, sterilized milk and chicken meat). The detection limit of the real-time PCR assay however remained unaffected in the presence of 10⁸ CFU/ml Salmonella typhimurium in another analysis. A total of 92 food samples were analyzed for the presence of Cronobacter, out of which two pork samples were found as positive by real-time PCR, whereas only one was detected by the ISO standard method. The adjusted real-time PCR assay can be adopted to rapidly detect Cronobacter spp. in food samples with high specificity and sensitivity, and can prevent false negative results by using the IAC.     

Development of a multiplex real-time PCR method for simultaneous detection of Vibrio parahaemolyticus,Listeria monocytogenes and Salmonella spp. in raw shrimp

Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. are important pathogens contaminating seafood in China. In this study, we developed an efficient multiplex real-time PCR for the simultaneous detection of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in raw shrimp without a prior enrichment step. In a test using 28 target and non-target strains only the targets were detected and two calibration curves, for pure cultures and artificially contaminated samples, were used to evaluate the efficiencies of this method. Amplification efficiencies of this multiplex real-time PCR were excellent in pure cultures and artificially contaminated shrimps. The limits of detection in artificially contaminated shrimps were 112 CFU/g for V. parahaemolyticus, 158 CFU/g for L. monocytogenes and 103 CFU/g for Salmonella spp. We validated this multiplex real-time PCR method on 48 commercial samples and the results were comparable to standard culture methods. This efficient multiplex real-time PCR, where each test takes only 50 min after DNA extraction, is a useful tool for high-throughput surveillance of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in seafood products.     

Investigation on prevalence of Listeria spp. and Listeria monocytogenes in animal-derived foods by multiplex PCR assay targeting novel genes

Chilled and frozen animal-derived food can be contaminated by Listeria spp., emerging foodborne pathogens in food industry. The objective of this study was to mine novel target genes by comparative genomics approach for multiplex PCR detection and differentiation of Listeria monocytogenes and other Listeria spp. in food. Multiplex PCR assay targeting the genetic markers LMOf2365_2721, AX25_00730, lin1814, int, lwe1673, and Oxidoreductase gene, resulted in the amplification of DNA fragments of 583 bp, 703 bp, 421 bp, 994 bp, 345 bp, and 201 bp from L. monocytogenes, Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi, respectively. The detection limits of the multiplex assays were as low as 89 fg/μL genomic DNA and 910 CFU/mL of bacterial culture. The prevalence of Listeria spp. was determined using the developed multiplex PCR assay and standard microbiological method in a total of 200 food samples collected from different supermarkets and traditional agri-product markets in Nanjing, China. A total of 28 samples were found to be positive for the presence of Listeria, including 10.9% (6/55) of livestock meat samples, 22% (11/50) of poultry samples, 15% (6/40) of shellfish samples, 13.3% (4/30) of octopus samples and 4% (1/25) of freshwater fish samples. Of these, 13 isolates were classified as L. monocytogenes, 11 were classified as L. innocua, 2 were classified as L. ivanovii and 3 were classified as L. welshimeri. These results demonstrate that the multiplex PCR assay based on novel target genes is able to rapidly detect the Listeria spp. in 12 h with high accuracy and sensitivity, which may be used in the future for detection of Listeria spp. in animal-derived food products.     

Detection of lupine (Lupinus spp.) DNA in processed foods using real-time PCR

Materials from lupine (Lupinus spp.) plants are used as food ingredients. Even small amounts can evoke allergic reactions in sensitized individuals. The applicability of a recently developed real-time PCR method to the sensitive and specific detection of lupine DNA in processed foods was examined. Using the preparation of pizza as a model, changes in the amplification efficiency and in the limit of detection arising from various processing steps such as freezing and baking were observed. For the starting flour the same limit of detection (0.1 mg/kg) as in raw foods was determined. Freezing of the dough resulted in increased cycle threshold values compared to the flour and the prepared dough. In the baked pizza the detection limit was 1 mg/kg, thus demonstrating the suitability of the method for the detection of lupine DNA in processed foods.     

Occurrence and Co-occurrence of Alternaria toxins in tomato-based products collected in China

A total of 174 tomato-based products collected in China in 2021 were analyzed for the natural occurrence of four Alternaria toxins: alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), and tentoxin (TEN) by ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry. The results showed that TeA was the predominant toxin (average: 37.6 μg/kg and maximum: 770.1 μg/kg). The positive samples showed a decreasing order of average, maximum, and median concentrations of the four Alternaria toxins: TeA > AOH > AME > TEN. There was a significant correlation between AOH and AME in all analyzed samples (r = 0.785, p < 0.05). Almost 95% of samples were with Alternaria toxins, specifically, 16.1% with one kind of toxin, 24.7% with two, 12.1% with three, and 41.4% with four toxins. Higher concentrations of toxins were in dried tomatoes compared to other types of tomato-based products. Moreover, there was no significant difference in Alternaria toxins concentrations of products made in China, Europe, America, Asia, and Australia. This survey revealed that the co-contamination of the four Alternaria toxins in commercially tomato-based products was very common. Ongoing surveillance of tomato-based products from China is necessary to gather more data, so specific regulations for Alternaria toxins in tomato-based products can be set in the future.     

Validation of a Real-time PCR assay for fast and sensitive quantification of Brucella spp. in water buffalo milk

Water buffalo milk and derived dairy products, including mozzarella cheese, represent a possible source of Brucella contamination for consumers. Brucella is a severe pathogen for human health even at low concentrations. It is therefore fundamental to develop an assay that is faster and more sensitive than the traditional bacterial culturing method for the detection of the pathogen in the food matrix. We designed a Real-time PCR assay able to detect as low as 1 CFU/ml of Brucella spp. in water (80% probability) and 3 CFU/ml of Brucella spp. in buffalo milk (50% probability) in less than 3 h without any enrichment step. The assay was validated by calculating specificity, sensitivity, detection limit, precision, PCR efficiency, DNA extraction efficiency and food matrix inhibition. When this method was employed to detect and quantify Brucella spp. in 109 buffalo milk samples, the assay demonstrated a higher sensitivity in comparison to bacteriological analysis (27 positive samples and 2 positive samples, respectively).     

Development of a real time PCR assay for detection of allergenic trace amounts of peanut (Arachis hypogaea) in processed foods

Peanut allergic reactions can result from the ingestion of even very small quantities of peanut and represent a severe threat to the health of sensitized individuals. In order to protect the allergic consumer, efficient and reliable methods are required for the detection of allergenic ingredients. For this purpose, we have developed a TaqMan real-time PCR method for specific detection of peanut in commercial food products. The assay uses two genetic makers in the Arah2 (125 bp) and ITS1 (90 bp) gene respectively, and TaqMan probes. The nuclear 18S rRNA gene was employed as a positive amplification control. Results obtained on sensitivity of both Arah2 and ITS primers with mixtures spiked with different concentrations of the target showed detection levels of 10 and 0.1 ppm, respectively. The applicability of the real-time PCR protocol to detect the presence of peanut DNA in commercial food products was determined through analysis of 123 different commercial products with the ITS primers which aimed higher sensitivity than Arah2 primer pair. The performance of the real-time PCR proposed herein allows a highly sensitive detection of peanut DNA in all the different food products, which declared to contain peanut material as well as in others were the presence of peanut or its traces wasn't declared in the labeling.     

Molecular screening and characterization of Shiga toxin-producing Escherichia coli in retail foods

Shiga toxin-producing Escherichia coli (STEC) strains are one of the most important recently emerged groups of food borne pathogens. This study investigated the prevalence of molecular markers for STEC and characteristics of E. coli O157 isolates from foods sold at retail markets in Wuhan, China. A total of 489 samples (350 meat products and 139 raw vegetables) were purchased from 22 large scale markets between July of 2011 and September of 2013. The meat samples consisted of frozen chicken products, raw pork, raw beef, frozen fish products and processed duck products. The raw vegetable samples consisted of lettuce, bok choy, radish, spinach, cucumber, and tomato. Shiga toxin genes (stx1 and stx2) and an O-group marker of the seven main pathogenic STEC serogroups (O157, O26, O45, O103, O111, O121, and O145) were detected in the samples by using PCR. 100% agreement was obtained between the results of the PCR targeting for wzy_O157 and the PCR targeting for rfbE_O157 gene. The result demonstrated that PCR assay targeting for wzy_O157 gene can be employed as an effective screening method for E. coli O157 in food sample. In the study, E. coli O157 and non-O157 STEC were detected in 55 (11.2%) and 75 (15.3%) samples by PCR screening, respectively. There was significant difference in the occurrence of STEC contamination between supermarkets (19/127, 15.0%) and open markets (111/362, 30.7%) (P < 0.05). Out of 489 samples, 5 samples carried O45, 1 sample carried O145 and 1 sample carried O111. Markers for O103, O26 and O121 were not detected. This result differed from other reports. Immunomagnetic separation based cultivation technique was used to isolate E. coli O157 from 27 food samples collected in 2013. Finally 7 E. coli O157 isolates were obtained. Among the 7 isolates, the prevalent stx genotype was stx_1a and stx_2a. Four E. coli O157 strains exhibited toxic effects on Vero cells, while 3 isolates had no detectable cytotoxicity effects even though they contained stx genes. All E. coli O157 isolates were sensitive to the 12 antimicrobials tested except for roxithromycin. There are some inconsistencies between the PCR screening and culture results. Characteristics of STEC isolates should be evaluated and considered for monitoring STEC contamination in foods.     

Detection of Listeria monocytogenes using a commercial PCR kit and different DNA extraction methods

The aim of our work was to evaluate a new commercial test kit for the detection of Listeria monocytogenes by PCR, using different DNA extraction methods. Food samples (pork sausage and “mozzarella” cheese) were spiked with known concentrations of L. monocytogenes and culture-enriched for 24 h. DNA extracted using three commercial kits and two standard methods, was amplified in species-specific PCR employing a L. monocytogenes PCR Detection Kit (Diatheva). The PCR-based method proved to be a reliable means of detecting the pathogen in food samples independently from the extraction procedure used, even for a contamination cell number of 1 cfu/g before culture enrichment. The molecular assay, showing perfect agreement with standard microbiological tests and a considerably shortened analysis time, provides a sensitive and rapid alternative for applications in the testing of foods for microbiological contamination, and highlights the potential of PCR technology in routine food control.     

Quantification of Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 in non-spiked food products and evaluation of real-time PCR as a diagnostic tool in routine food analysis

Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp. are foodborne pathogens frequently associated with foods such as poultry, ready-to-eat products, fruits and vegetables. PCR-based procedures are rapid, sensitive and accurate; in particular, real-time PCR (qPCR), which besides being an automated high-throughput technique, allows quantification of foodborne pathogens. In the present work, qPCR-based methods were applied for the quantitative detection of E. coli O157:H7, Salmonella spp. and L. monocytogenes in a total of 306 non-spiked food samples in a study carried out in two laboratories simultaneously. qPCR allowed the detection of the three pathogens in around 20% of the analyzed samples for each pathogen. Quantification results revealed the presence of the three pathogens mostly at levels between 10² and 10⁴ cells/g. Besides quantification, the qPCR results (presence/absence) were compared with those of the standard mini-VIDAS system. In order to determine which were the “true” positive samples, conventional PCR was carried out after the corresponding enrichment for each pathogen. These results were considered as the gold standard for further analysis. The statistical analysis of global data recording the presence of E. coli O157:H7 and L. monocytogenes, together with data previously obtained for Salmonella spp., revealed that qPCR outperformed the mini-VIDAS procedures, in terms of both time and accuracy. Thus, these results proved qPCR to be useful as a rapid diagnostic test for the direct detection of pathogens in food, without the need for enrichment steps.     

Using the PCR–RFLP method to identify the species of different processed products of billfish meats

A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method had been developed for the detection of five billfish species Xiphias gladius, Makaira nigricans, M. indica, Istiophorus platypterus and Tetrapturus audax in raw, frozen and heat-treated meats. The primers L-CYTBF and H-CYTBF were designed in the mitochondrial cytochrome b (cytb) gene and the molecular weight of amplified fragment was 348 bp and amplified the fragment from processed billfish meats. The results obtained from the BsaJI, Cac8I and HpaII enzymes digestion could be used to distinguish the five billfish species in frozen and heat-treated meats. Using the PCR–RFLP method, species of 10 commercial samples including raw fish fillets, frozen fish meats and fried fish meats could be identified. It was determined that two commercial samples of billfish products were not made from billfish. The method is sensitive, rapid and valid to detect fraudulent billfish products substituted from cheaper fish.     

Prevalence and pattern of antibiotic resistance of Campylobacter spp. in poultry meat in Southern Italy

Campylobacter spp. is one of the most common cause of sporadic human foodborne illness in industrialized countries, and concern has been raised by the emergence of strains showing antibiotic resistance. The aim of this study was to investigate prevalence of Campylobacter spp. and pattern of antibiotic resistance in samples of poultry raw meat sold in retail outlets in Southern Italy.A total of 208 samples of chicken and turkey raw meat collected from randomly selected retail butchers' shops in Catanzaro (Italy) were processed for the presence of Campylobacter spp.Campylobacter spp. contaminated 43 samples of poultry product. The most frequent isolates were Campylobacter coli (34.9%) and Campylobacter jejuni (32.6%). The lowest resistance was found for gentamycin (27.9%) and cloramphenicol (32.6%). We also investigated prevalence of sensitivity to the most important antibiotics and we found that only 20.9% isolates were sensible to both ciprofloxacin and erythromycin.Our findings revealed a reduction in the overall contamination by Campylobacter spp. in raw meat. However, resistant and particularly multi-resistant strains were alarmingly spread, representing an increasing phenomenon that demands enforced interventions at multidisciplinary levels.     

Isolation,identification and antimicrobial resistance of Cronobacter spp. isolated from various foods in China

Cronobacter spp. are important foodborne pathogens that can cause severe diseases such as meningitis, sepsis, and necrotizing enterocolitis in neonates. In this study, 195 food samples, including cereals, cereal products, powdered infant formula (PIF), infant food formula, herbs, spices, vegetables, and fruits, were analyzed for the presence of Cronobacter spp. by culture-based method. The presumptive isolates were further confirmed by targeting the 16S rDNA gene using PCR. Out of 195 samples, 13 samples (6.7%) were positive for Cronobacter species. 12 of 85 cereal and cereal products (14.1%), and 1 of 22 herbs and spices (4.5%) were contaminated. In contrast, no Cronobacter was detected in commercial powdered infant formula, infant food formula, vegetables, or fruits. Alignment of 16S rRNA gene sequences showed that 13 isolates was most closely related to the genus Cronobacter. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis revealed that Cronobacter sakazakii was the only Cronobacter species isolated from various food samples. The antimicrobial susceptibility of 13 Cronobacter isolates was determined by the standard disk diffusion method. All isolated strains, except one resistant to ampicillin, were sensitive or displayed intermediate susceptibility to the 10 antimicrobial agents investigated. No multiple drug resistance was observed.     

Detection of verocytotoxin-producing Escherichia coli (VTEC) in minced beef and raw milk by colony blot hybridization

Verocytotoxin-producing Escherichia coli (VTEC) are foodborne pathogens that cause outbreaks linked to consumption of meat and raw milk. In this note the authors report results obtained from a survey conducted on minced beef and raw bovine milk samples using a Multiplex PCR (M-PCR) for the detection of eae, stx₁, stx₂ and hlyA genes as a screening step followed by a colony blot hybridization (CBH) technique for the isolation of the VTEC. Of 100 minced beef and 123 raw milk samples, 13 (13%) and 7 (5.7%) were positive in the M-PCR and among these 9 and 3 strains were isolated using CBH, respectively. All isolates showed the presence of the stx₂ gene, single or in association with the other investigated genes. None of the isolates belonged to the O157, O26, O91, O103, O111 and O145 serogroups. The study showed that the use of M-PCR for the screening of samples coupled with a sensitive and specific detection technique, could improve the possibility of detection of VTEC strains in foods. Moreover, the presence of VTEC in minced beef and in raw milk confirms their important role as putative vehicles of infection to humans. Stringent control of these foodstuffs is essential for food safety purposes.     

The application of Bar-HRM (Barcode DNA-High Resolution Melting) analysis for authenticity testing and quantitative detection of bean crops (Leguminosae) without prior DNA purification

The Leguminoseae family includes several economically important genera like legumes. Legumes are the second most important agricultural family after cereals in terms of harvested area and production. Legumes are key elements in the Mediterranean diet and as winter annuals with nitrogen fixing capacity, they are extremely important for sustainable agriculture worldwide.We report here the application of the Bar-HRM (Barcode DNA High Resolution Melting) analysis method with the universal plant DNA barcoding region trnL which allowed the identification, adulteration and quantification of major Greek and Mediterranean in general bean species. Bar-HRM detected Lupinus spp. adulterants in Glycine max flour as low as 1:100. Moreover, the method was coupled with the rapid Phire PCR kit that does not require prior DNA purification. This makes the method a very fast and effective tool for barcoding Legumes and particularly for the crops examined not only for their authenticity but for quantitative detection of purity of their seeds or their processed food and feed products.