Recent developments in the use of viability dyes and quantitative PCR in the food microbiology field.

The increase in foodborne outbreaks highlights the need for rapid, sensitive and specific methods for food safety monitoring, enabling specific detection and quantification of viable foodborne pathogens. Real-time PCR (qPCR) combined with the use of viability dyes, recently introduced, fulfils all these requirements. The strategy relies on the use of DNA-binding molecules such as propidium monoazide (PMA) or ethidium monoazide (EMA) as sample pretreatment previous to the qPCR. These molecules permeate only membrane-compromised cells and have successfully been applied for different types of foodborne pathogens, including bacteria and viruses. Moreover, those dyes have been explored to monitor different food manufacturing processes as an alternative to classical cultural methods. In this review, state-of-the-art information regarding viability PCR (v-PCR) is compiled.

Application of propidium monoazide-qPCR to evaluate the ultrasonic inactivation of Escherichia coli O157:H7 in fresh-cut vegetable wash water.

The efficacy of sanitizing technologies in produce or in vegetable wash water is generally evaluated by plate count in selective media. This procedure is time consuming and can lead to misinterpretations because environmental conditions and sanitizing processes may affect bacterial growth or culturable capability. Thus, the aim of this study was to determine the applicability of a propidium monoazide real-time PCR (PMA-qPCR) method to monitor the inactivation by ultrasound treatment of foodborne bacteria in fresh-cut vegetable wash water. To this aim, lettuce wash water was artificially inoculated with Escherichia coli O157:H7 (106 CFU/mL) and treated by means of a continuous ultrasonic irradiation with a power density of 0.280 kW/L. Quantification data obtained by PMA-qPCR and plate counts were statistically similar during the viability reduction of 99.996% which corresponds to 4.4 log reductions. Further reductions of E. coli O157:H7 were not detected by the PMA-qPCR method due to the limit of detection of this technique (20 CFU/mL). Inactivation data obtained by both techniques successfully fitted a linear model, giving no significant differences in kinetic parameters. These results indicate that the PMA-qPCR method is a suitable technique for evaluating ultrasonic disinfection of vegetable wash water, being able to distinguish between live and dead bacteria.

A single method for recovery and concentration of enteric viruses and bacteria from fresh-cut vegetables.

Fresh-cut vegetables are prone to be contaminated with foodborne pathogens during growth, harvest, transport and further processing and handling. As most of these products are generally eaten raw or mildly treated, there is an increase in the number of outbreaks caused by viruses and bacteria associated with fresh vegetables. Foodborne pathogens are usually present at very low levels and have to be concentrated (i.e. viruses) or enriched (i.e. bacteria) to enhance their detection. With this aim, a rapid concentration method has been developed for the simultaneous recovery of hepatitis A virus (HAV), norovirus (NV), murine norovirus (MNV) as a surrogate for NV, Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enterica. Initial experiments focused on evaluating the elution conditions suitable for virus release from vegetables. Finally, elution with buffered peptone water (BPW), using a Pulsifier, and concentration by polyethylene glycol (PEG) precipitation were the methods selected for the elution and concentration of both, enteric viruses and bacteria, from three different types of fresh-cut vegetables by quantitative PCR (qPCR) using specific primers. The average recoveries from inoculated parsley, spinach and salad, were ca. 9.2%, 43.5%, and 20.7% for NV, MNV, and HAV, respectively. Detection limits were 132 RT-PCR units (PCRU), 1.5 50% tissue culture infectious dose (TCID50), and 6.6 TCID50 for NV, MNV, and HAV, respectively. This protocol resulted in average recoveries of 57.4%, 64.5% and 64.6% in three vegetables for E. coli O157:H7, L. monocytogenes and Salmonella with corresponding detection limits of 10³, 10² and 10³ CFU/g, respectively. Based on these results, it can be concluded that the procedure herein is suitable to recover, detect and quantify enteric viruses and foodborne pathogenic bacteria within 5 h and can be applied for the simultaneous detection of both types of foodborne pathogens in fresh-cut vegetables.

Evaluation of yogurt and various beverages as carriers of lactic acid bacteria producing 2-branched (1,3)-ß-D-glucan.

Probiotic cultures are increasingly being incorporated into a wide variety of food products. Although lactobacilli and bifidobacteria are the most frequently used, other lactic acid bacteria (LAB) have been reported to be potential probiotics. Of these, the cider isolates Pediococccus parvulus (strains 2.6 and CUPV22) and Lactobacillus suebicus CUPV221 produce a 2-branched (1,3)-ß-d-glucan exopolysaccharide that decreases serum cholesterol levels and affects the activation of human macrophages. For this reason, these 3 strains were incorporated into yogurt, orange juice, and 2 juice-milk beverages to evaluate the effect of the food matrix on the resistance of these strains to simulated gastrointestinal tract conditions. Our results showed that incorporation of the LAB did not significantly affect the physical and rheological properties of the food matrices tested. When incorporated in yogurt, LAB strains population decreased by 2 to 3 log orders of magnitude during the shelf life of the product (28 d). However, no significant decrease was observed in the juice and juice-milk beverages during the same storage period, except for Lb. suebicus, whose viability decreased by 3 log orders of magnitude. When strains were subjected to gastrointestinal tract conditions, a decrease in the survival was observed at the lower pH (1.8). However, incorporation of these LAB strains into orange juice increases their resistance to lower pH conditions, thus improving survival to gastrointestinal stress. Moreover, a protective effect was observed for P. parvulus CUPV22 and 2.6 to gastric stress in juice-milk beverages and to gastrointestinal stress in yogurt. Lactobacillus suebicus CUPV221 did not survive when incorporated into yogurt and juice-milk beverage.

Simultaneous detection of the main black aspergilli responsible for ochratoxin A (OTA) contamination in grapes by multiplex real-time polymerase chain reaction.

This paper reports a duplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of members of the Aspergillus niger aggregate and A. carbonarius, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the beta-ketosynthase and the acyl transferase domains of the poliketide synthase of A. carbonarius and the A. niger aggregate, respectively. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic T(m)-values demonstrating the specific, efficient and balanced amplification of the two PCR fragments. Subsequently, a TaqMan real-time PCR approach was settled, using 6-carboxy-fluorescein group (FAM) and VIC-labelled specific probes for automated detection. Results indicated no differences in sensitivity when using either the two sets of primers and probes in separate or in the same reaction. However, when both targets are in very different amounts, there is a preferential amplification of the target which is in more concentration. CT-values obtained in the presence of grape DNA were very similar to those observed when only fungal purified DNA was present, indicating that the grape DNA does not interfere in the real-time PCR reaction. This procedure provides a fast and accurate tool to monitor, in a single reaction, the presence of OTA-producing species in grapes which, to some extent, will facilitate OTA contamination surveys to guarantee food safety in the wine industry.

Molecular characterization of the black Aspergillus isolates responsible for ochratoxin A contamination in grapes and wine in relation to taxonomy of Aspergillus section Nigri.

This work examines ochratoxigenic mycobiota in grapes by ap-PCR analysis sequence analysis of the ITS and IGS regions and ability to produce OTA. A comparison was also made with many reference strains of Aspergillus section Nigri. Based on ap-PCR profiles, derived from two microsatellite primers, three main groups were obtained by UPGMA cluster analysis corresponding to A. carbonarius, A. niger and A. tubingensis. The cophenetic correlation values corresponding to ap-PCR UPGMA analysis revealed a higher genetic variability in A. niger and A. tubingensis than in A. carbonarius. In addition, no genotypical differences could be established between OTA producers and nonproducers in all species analysed. Phylogenetic relationships inferred from ITS and IGS sequences are, mostly, congruent with earlier works. A. niger and A. tubingensis strains were closely related, but not identical, and they clustered into two distinct groups within the A. niger aggregate. Sequence analysis also showed genetic divergences between strains of A. foetidus and the rest of the Aspergillus section Nigri. Additionally, the phylogenetic analysis was consistent in separating a new group of ochratoxigenic strains, frequently isolated from grapes, named A. tubingensis-like. All strains of A. carbonarius analysed by sequence analysis had identical ITS and IGS sequences confirming the lack of significant genetic variability within this important ochratoxigenic species.

Comparison of four commercial DNA extraction kits for PCR detection of Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, and Staphylococcus aureus in fresh, minimally processed vegetables.

Four commercial DNA extraction methods, PrepMan Ultra (Applied Biosystems), InstaGene Matrix (BioRad), DNeasy Tissue kit (Qiagen), and UltraClean (MoBio), were tested for PCR detection of Listeria monocytogenes, Escherichia coli O157: H7, Salmonella, and Staphylococcus aureus in fresh, minimally processed vegetables. For comparative purposes, sensitivity assays with specific PCRs were carried out after DNA extraction with the four methods in green pepper, broccoli, and onion artificially inoculated with the four pathogens separately. As confirmed by statistical analysis, the DNeasy Tissue kit rendered the highest sensitivity values in the three matrices assayed for Salmonella, L. monocytogenes, and E. coli O157:H7 and in onion for S. aureus. Despite being the most expensive of the methods compared, the DNeasy Tissue Kit can be successfully applied for any of the four most commonly studied pathogens, thus saving time and overall reducing the cost of the analysis.

Potential microbial risk factors related to soil amendments and irrigation water of potato crops.

AIMS: This study assesses the potential microbial risk factors related to the use of soil amendments and irrigation water on potato crops, cultivated in one traditional and two intensive farms during two harvest seasons. METHODS AND RESULTS: The natural microbiota and potentially pathogenic micro-organisms were evaluated in the soil amendment, irrigation water, soil and produce. Uncomposted amendments and residual and creek water samples showed the highest microbial counts. The microbial load of potatoes harvested in spring was similar among the tested farms despite the diverse microbial levels of Listeria spp. and faecal coliforms in the potential risk sources. However, differences in total coliform load of potato were found between farms cultivated in the autumn. Immunochromatographic rapid tests and the BAM's reference method (Bacteriological Analytical Manual; AOAC International) were used to detect Escherichia coli O157:H7 from the potential risk sources and produce. Confirmation of the positive results by polymerase chain reaction procedures showed that the immunochromatographic assay was not reliable as it led to false-positive results. CONCLUSIONS: The potentially pathogenic micro-organisms of soil amendment, irrigation water and soil samples changed with the harvest seasons and the use of different agricultural practices. However, the microbial load of the produce was not always influenced by these risk sources. Improvements in environmental sample preparation are needed to avoid interferences in the use of immunochromatographic rapid tests. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential microbial risk sources of fresh produce should be regularly controlled using reliable detection methods to guarantee their microbial safety.

Lactic acid bacteria associated with vacuum-packed cooked meat product spoilage: population analysis by rDNA-based methods.

AIM: To determine the lactic acid bacteria (LAB) implicated in bloating spoilage of vacuum-packed and refrigerated meat products. METHODS AND RESULTS: A total of 18 samples corresponding to four types of meat products, with and without spoilage symptoms, were studied. In all, 387 colonies growing on de Man, Rogosa and Sharpe, yeast glucose lactose peptone and trypticase soy yeast extract plates were identified by internal spacer region (ISR), ISR-restriction fragment length polymorphism and rapid amplified ribosomal DNA restriction analysis profiles as Lactobacillus (37%), Leuconostoc (43%), Carnobacterium (11%), Enterococcus (4%) and Lactococcus (2%). Leuconostoc mesenteroides dominated the microbial population of spoiled products and was always present at the moment bloating occurred. Lactobacillus sakei, Lactobacillus plantarum and Lactobacillus curvatus were found in decreasing order of abundance. The analysis of two meat products, 'morcilla' and 'fiambre de magro adobado' obtained from production lines revealed a common succession pattern in LAB populations in both products and showed that Leuc. mesenteroides became the main species during storage, despite being below the detection level of culture methods after packing. CONCLUSIONS: Our results pointed to Leuc. mesenteroides as the main species responsible for bloating spoilage in vacuum-packed meat products. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevention of bloating spoilage in vacuum-packed cooked meat products requires the sensitive detection of Leuc. mesenteroides (i.e. by PCR).