Improving efficiency of viability-qPCR for selective detection of infectious HAV in food and water samples.

AIM: To improve the efficacy of intercalating dyes to distinguishing between infectious and inactivated hepatitis A virus (HAV) in food. METHODS AND RESULTS: Different intercalating dyes were evaluated for the discrimination between infectious and thermally inactivated HAV suspensions combining with the RT-qPCR proposed in the ISO 15216. Among them, PMAxx was the best dye in removing the RT-qPCR signal from inactivated HAV. Applied to lettuce and spinach, PMAxx-Triton pretreatment resulted in complete removal of the RT-qPCR signal from inactivated HAV. Likewise, this study demonstrates that this pretreatment is suitable for the discrimination of inactivated HAV in shellfish without further sample dilution. In mussels and oysters, the developed viability RT-qPCR method reduced the signal of inactivated HAV between 1·7 and 2·2 logs at high inoculation level, and signal was completely removed at low inoculation level. CONCLUSIONS: This study showed that the use of PMAxx is an important improvement to assess HAV infectivity by RT-qPCR. It was shown that PMAxx-Triton pretreatment is suitable for the analysis of infectious HAV in complex food samples such as vegetables and shellfish. SIGNIFICANCE AND IMPACT OF THE STUDY: The PMAxx-Triton pretreatment can be easily incorporated to the ISO norm for infectious virus detection.

Irrigating Lettuce with Wastewater Effluent: Does Disinfection with Chlorine Dioxide Inactivate Viruses?

Reclaimed water obtained from urban wastewater is currently being used as irrigation water in water-scarce regions in Spain. However, wastewater can contain enteric viruses that water reclamation treatment cannot remove or inactivate completely. In the present study, greenhouse-grown baby lettuce ( L.) was irrigated with secondary treatment effluent from a wastewater treatment plant untreated and treated using chlorine dioxide (ClO). The effect of ClO treatment on the physicochemical characteristics and the presence of enteric viruses in irrigation water and lettuce was assessed. The presence of human noroviruses genogroups I and II (NoV GI and NoV GII), and human astroviruses (HAstV), was analyzed by real-time polymerase chain reaction (RT-qPCR). Additionally, to check for the loss of infectivity induced by the disinfection treatment, positive samples were re-analyzed after pretreatment with the intercalating dye PMAxx before RNA extraction and RT-qPCR. There were no significant differences in the proportion of positive samples and the concentration of enteric viruses between treated and untreated reclaimed water without PMAxx pretreatment ( > 0.05). A significantly lower concentration of NoV GI was detected in ClO-treated water when samples were pretreated with PMAxx ( < 0.05), indicating that inactivation was due to the disinfection treatment. Laboratory-scale validation tests indicated the suitability of PMAxx-RT-qPCR for discrimination between potentially infectious and ClO-damaged viruses. Although the applied ClO treatment was not able to significantly reduce the enteric virus load of the secondary effluent from the wastewater treatment plant, none of the lettuce samples analyzed ( = 36) was positive for the presence of NoV or HAstV.

Effect of green tea extract on enteric viruses and its application as natural sanitizer.

In this work, the effect of green tea extract (GTE) was assessed against murine norovirus (MNV) and hepatitis A virus (HAV) at different temperatures, exposure times and pH conditions. Initially, GTE at 0.5 and 5 mg/ml were individually mixed with each virus at 5 log TCID50/ml and incubated 2 h at 37 °C at different pHs (from 5.5 to 8.5). GTE affected both viruses depending on pH with higher reductions observed in alkaline conditions. Secondly, different concentrations of GTE (0.5 and 5 mg/ml) were mixed with viral suspensions and incubated for 2 or 16 h at 4, 25 and 37 °C at pH 7.2. A concentration-, temperature- and exposure time-dependent response was showed by GTE in suspension tests, where complete inactivation was achieved after overnight exposure at 37 °C for both viruses and also at 25 °C for HAV. In addition, antiviral effect of GTE proved efficient in the surface disinfection tests since 1.5 log reduction and complete inactivation were recorded for MNV and HAV on stainless steel and glass surfaces treated with 10 mg/ml GTE for 30 min, analyzed in accordance with ISO 13697:2001. GTE was also evaluated as a natural disinfectant of produce, showing 10 mg/ml GTE reduced MNV and HAV titers in lettuce and spinach by more than 1.5 log after 30 min treatment. The results show a potential of GTE as natural disinfectant able to limit enteric viral (cross-)contaminations conveyed by food and food-contact surfaces.

Occurrence of enteric viruses in reclaimed and surface irrigation water: relationship with microbiological and physicochemical indicators.

AIMS: To assess the prevalence of enteric viruses in different irrigation water sources and in the irrigated produce, and the possible links with microbiological and physicochemical water characteristics. METHODS AND RESULTS: The prevalence and levels of Escherichia coli, Norovirus (NoV) genogroup I (GI) and II (GII), as well as Hepatitis A virus were assessed in three types of water: surface water (surface-W), reclaimed water subjected to secondary treatment (secondary-W) and reclaimed water subjected to tertiary treatment (tertiary-W), as well as in zucchini irrigated with these irrigation water sources. Chemical oxygen demand (COD), turbidity, total suspended solids, alkalinity and maximum filterable volume (MFV) were also measured in the water. Higher prevalence of NoV in secondary-W (GI 100%, GII 55·6%) and tertiary-W (GI 91·7%, GII 66·7%) compared with surface-W (GI 58·4%, GII 22·2%) was observed. Nov GI showed positive correlation with E. coli (Spearman's correlation coefficient = 0·68, P < 0·01), and with some physicochemical parameters such as COD (0·52, P < 0·01), turbidity (0·52, P < 0·01) and MFV (0·54, P < 0·01). Escherichia coli and enteric viruses were not detected in zucchini. CONCLUSION: There is a potential risk of contamination of crops with NoV when reclaimed water is used for irrigation. SIGNIFICANCE AND IMPACT OF THE STUDY: Increase the knowledge on the prevalence of enteric viruses in different irrigation water sources, and its consequences for fresh produce safety.

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.

Evaluation of phenotypic and PCR-based approaches for routine analysis of Bacillus cereus group foodborne isolates.

Identification of Bacillus cereus sensu stricto is a challenge for the food industry since it is being increasingly reported as implicated in many foodborne outbreaks. So far no conclusive microbiological or biochemical traits have been described for their specific differentiation. Here a polyphasic approach aiming at identification of new isolates is presented. It was conducted on a total of 75 strains, 59 Bacillus cereus group (29 reference strains and 30 food and environmental isolates) and 16 other Bacillus species. It includes biochemical traits (API 50CH and API 20E) and genetic profiles: PCR amplification of the internal spacer region (ISR) between 23S and 16S rRNA genes (ISR-PCR), randomly amplified polymorphic DNA (RAPD-PCR) with three universal primers (M13, T3, and T7), and PCR amplification using specific primers directed to genes encoding hemolysin BL (hbl), cytotoxin K (cytK) and cereulide (ces). As expected, PCR-enterotoxin profiles revealed the toxigenic potential of strains within the B. cereus group irrespective of the species. Cluster analysis combining the three RAPD fingerprints (RAPD-M13, RAPD-T3 and RAPD-T7) allowed almost a complete separation of strains within the B. cereus group. As a result, the ISR-PCR profile is proposed for the rapid assignation of isolates to B. cereus group with the advantage over the API profile of being a specific and culture-independent technique. Following, differentiation at species level can be obtained by RAPD profiles analysis.

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.

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.

Allergic reaction after ingestion of orange blossom pollen.

A 31-year-old atopic subject with allergic rhinoconjunctivitis with sensitization to several pollens, presented with urticaria and angioedema after ingestion of orange blossom pollen (Citrus sinensis). A positive skin prick test for orange blossom pollen extract (BIAL-Aristegui, Bilbao, Spain) at a concentration of 1.2 mg/ml was obtained. Serum specific IgE antibodies to orange blossom pollen were shown (Unicap Pharmacia System, Uppsala, Sweeden). A conjunctival provocation test was negative with orange blossom pollen extract dilutions of 1:1000, 1:100 and 1:10 w/v. We describe a patient with an IgE-mediated reaction caused by hypersensitivity to orange blossom pollen. Although the pollen is an aeroallergen and the way of sensitization and entrance is the airway, and therefore the symptoms appear in this location, when contact is through some other route such as the digestive tract, it is also able to sensitize reporting urticaria and angioedema like in our case, instead of respiratory symptoms.

Curcumin-Mediated Photodynamic Inactivation of Norovirus Surrogates.

Photodynamic inactivation (PDI) is extensively used to inactivate different type of pathogens through the use of photosensitizers (PS). Curcumin has been identified as an excellent natural photosensitizer with some potential applications in the food industry. The aim of this study was to assess the antiviral activity of photoactivated curcumin on norovirus surrogates, feline calicivirus (FCV), and murine norovirus (MNV). Initially, different concentrations of curcumin (13.5-1358 µM) were individually mixed with each virus at titers of ca. 6-7 log TCID50/ml and photoactivated by LED blue light with light dose of 3 J/cm2. Results showed that photoactivated curcumin at 50 µg/mL reduced FCV titers by almost 5 log after incubation at 37 °C for 30 min. Lower antiviral activity (0.73 log TCID50/mL reduction) was reported for MNV. At room temperature, curcumin at 5 µg/mL reduced FCV titers by 1.75 log TCID50/mL. These results represent a step forward in improving food safety using photoactivated curcumin as an alternative natural additive to reduce viral contamination.

Evaluation of viability PCR performance for assessing norovirus infectivity in fresh-cut vegetables and irrigation water.

Norovirus (NoV) detection in food and water is mainly carried out by quantitative RT-PCR (RT-qPCR). The inability to differentiate between infectious and inactivated viruses and the resulting overestimation of viral targets is considered a major disadvantage of RT-qPCR. Initially, conventional photoactivatable dyes (i.e. propidium monoazide, PMA and ethidium monoazide, EMA) and newly developed ones (i.e. PMAxx and PEMAX) were evaluated for the discrimination between infectious and thermally inactivated NoV genogroup I (GI) and II (GII) suspensions. Results showed that PMAxx was the best photoactivatable dye to assess NoV infectivity. This procedure was further optimized in artificially inoculated lettuce. Pretreatment with 50µM PMAxx and 0.5% Triton X-100 (Triton) for 10min reduced the signal of thermally inactivated NoV by ca. 1.8 logs for both genogroups in lettuce concentrates. Additionally, this pretreatment reduced the signal of thermally inactivated NoV GI between 1.4 and 1.9 logs in spinach and romaine and lamb's lettuces and by >2 logs for NoV GII in romaine and lamb's lettuce samples. Moreover this pretreatment was satisfactorily applied to naturally-contaminated water samples with NoV GI and GII. Based on the obtained results this pretreatment has the potential to be integrated in routine diagnoses to improve the interpretation of positive NoV results obtained by RT-qPCR.

Efficacy of Cinnamaldehyde Against Enteric Viruses and Its Activity After Incorporation Into Biodegradable Multilayer Systems of Interest in Food Packaging.

Cinnamaldehyde (CNMA), an organic compound that gives cinnamon its flavor and odor, was investigated for its virucidal activity on norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), and hepatitis A virus (HAV). Initially, different concentrations of CNMA (0.1, 0.5 and 1 %) were individually mixed with each virus at titers of ca. 6-7 log10 TCID50/ml and incubated 2 h at 4 and 37 °C. CNMA was effective in reducing the titers of norovirus surrogates in a dose-dependent manner after 2 h at 37 °C, while HAV titers were reduced by 1 log10 after treatment with 1 % of CNMA. When incubation time was extended, HAV titers were reduced by 3.4 and 2.7 log10 after overnight incubation at 37 °C with 1 and 0.5 % of CNMA, respectively. Moreover, this paper analyzed, for the first time, the antiviral activity of adding an active electrospun interlayer based on zein and CNMA to a polyhydroxybutyrate packaging material (PHB) in a multilayer form. Biodegradable multilayer systems prepared with 2.60 mg/cm(2) (~9.7 %) of CNMA completely inactivated FCV according to ISO 22196:2011, while MNV titers were reduced by 2.75 log10. When the developed multilayer films were evaluated after one month of preparation or at 25 °C, the antiviral activity was reduced as compared to freshly prepared multilayer films evaluated at 37 °C. The results show the excellent potential of this system for food contact applications as well as for active packaging technologies in order to maintain or extend food quality and safety.

Evaluation of Natural Compounds of Plant Origin for Inactivation of Enteric Viruses.

Essential oils (EOs) and some of their main compounds have demonstrated extensive antimicrobial activity in a wide range of food spoilage or pathogenic fungi, yeast and bacteria. The aim of this study was to assess the antiviral activity of Zataria multiflora Boiss. (zataria) and Origanum vulgare (oregano) EOs on hepatitis A virus (HAV) and the effect of thymol, an active compound of Thymus vulgaris and oregano, on norovirus surrogates, feline calicivirus (FCV) and murine norovirus (MNV), and HAV. Initially, each virus at titers of ca. 6 log TCID50/ml was exposed to different concentrations of natural compounds and incubated for 2 h at 37 °C. Treatment with oregano and zataria EOs resulted in slight reductions on HAV infectivity with a maximum reduction of less than 0.5 log TCID50/ml at 0.1 % zataria EO. Thymol was effective in reducing the titers of norovirus surrogates in a dose-dependent manner. Concentrations of thymol at 0.5 and 1 % reduced FCV titers to undetectable levels, while for MNV, thymol at concentrations of 1 and 2 % resulted in reductions of 1.66 and 2.45 log TCID50/ml, respectively. However, for HAV, no effect was observed at any of the concentrations tested. These results improve the knowledge about the antiviral activity of EO and their compounds and their potential in food sanitation.

Application of viability PCR to discriminate the infectivity of hepatitis A virus in food samples.

Transmitted through the fecal-oral route, the hepatitis A virus (HAV) is acquired primarily through close personal contact and foodborne transmission. HAV detection in food is mainly carried out by quantitative RT-PCR (RT-qPCR). The discrimination of infectious and inactivated viruses remains a key obstacle when using RT-qPCR to quantify enteric viruses in food samples. Initially, viability dyes, propidium monoazide (PMA) and ethidium monoazide (EMA), were evaluated for the detection and quantification of infectious HAV in lettuce wash water. Results showed that PMA combined with 0.5% Triton X-100 (Triton) was the best pretreatment to assess HAV infectivity and completely eliminated the signal of thermally inactivated HAV in lettuce wash water. This procedure was further evaluated in artificially inoculated foods (at concentrations of ca. 6×10(4), 6×10(3) and 6×10(2)TCID50) including lettuce, parsley, spinach, cockles and coquina clams. The PMA-0.5% Triton pretreatment reduced the signal of thermally inactivated HAV between 0.5 and 2 logs, in lettuce and spinach concentrates. Moreover, this pretreatment reduced the signal of inactivated HAV by more than 1.5 logs, in parsley and ten-fold diluted shellfish samples inoculated at the lowest concentration. Overall, this pretreatment (50 µM PMA-0.5% Triton) significantly reduced the detection of thermally inactivated HAV, depending on the initial virus concentration and the food matrix.

The effect of carvacrol on enteric viruses.

Carvacrol, a monoterpenic phenol, is said to have extensive antimicrobial activity in a wide range of food spoilage or pathogenic fungi, yeast and bacteria. The aim of this study was to assess its antiviral activity on norovirus surrogates, feline calicivirus (FCV), murine norovirus (MNV), and hepatitis A virus (HAV), as well as its potential in food applications. Initially, different concentrations of carvacrol (0.25, 0.5, 1%) were individually mixed with each virus at titers of ca. 6-7 log TCID50/ml and incubated 2h at 37°C. Carvacrol at 0.5% completely inactivated the two norovirus surrogates, whereas 1% concentration was required to achieve ca. 1 log reduction of HAV. In lettuce wash water, carvacrol efficacy on MNV was dependent on the chemical oxygen demand (COD), with no effect over 300 ppm. A 4 log reduction in FCV infectivity was observed when 0.5% carvacrol was used to sanitize lettuce wash water, regardless of COD. Carvacrol was also evaluated as a natural disinfectant of produce, showing 1% carvacrol reduced inoculated NoV surrogates titers in lettuce by 1 log after 30 min contact. These results represent a step forward in improving food safety by using carvacrol as an alternative natural additive to reduce viral contamination in the fresh vegetable industry.

Siderophores and related outer membrane proteins produced by pseudomonads isolated from eels and freshwater.

A total of 46 environmental pseudomonads, together with six type strains, were examined for their siderophore-producing activity. All strains were able to grow under iron-limiting conditions, gave orange halos in the CAS agar assay, and produced hydroxamates, and some of them also produced phenolate-type compounds. Bioassays showed that all strains, except Pseudomonas aeruginosa, promoted growth of mutant strain Arthrobacter flavescens JG-9, deficient in hydroxamate production, and some of them promoted growth of Salmonella typhimurium enb-1, which requires enterobactin for growth. The presence of iron-regulated outer membrane proteins was observed, the molecular size of the main induced proteins ranged between 76 and 93 kDa.

Siderophore production by environmental strains of Salmonella species.

Iron uptake mechanisms were investigated in different species of Salmonella isolated from environmental waters. All strains examined were able to grow in the presence of high concentrations (10 mM) of the iron chelator EDDA. All strains excreted phenolate and hydroxamate siderophores, as assessed by bioassays and chemical tests. Bioassays with different indicator strains showed that all Salmonella strains can cross-feed other Enterobacteria, as well as mutants of Salmonella typhimurium deficient in the Enterobactin system, suggesting that this siderophore may be produced by the environmental Salmonella strains. The siderophore aerobactin may also be produced by one of the strains, according to the bioassays results. The same pattern of outer membrane proteins are synthesized under iron-limiting conditions in all species tested, which suggests a similarity of iron uptake systems in many species of Salmonella. This system could be also of great importance in the survival of these bacteria in natural waters, as well as in possible pathogenic mechanisms.

Electrocardiographic changes induced by insertion of an intrauterine device and other uterine manipulations.

A prospective study was carried out in normal women in order to investigate the effects of the following procedures on the electrocardiogram and blood pressure: (1) insertion of an intrauterine device (IUD)--the Lippes loop, Tcu-200, uterine progesterone system or Cu-7; (2) endometrial biopsy; and (3) uterine flushing. In groups in which a large IUD like the Lippes loop and/or a stiff IUD like the Cu-7 was inserted, the frequency of bradycardia was significantly higher than in any other groups. The severity of the bradycardia was similar in all of the groups and the frequency of this alteration was also similar among the nulliparous women or in those who complained of pain during the instrumentation, irrespective of the type of IUD inserted or the procedure carried out. No alarming modifications of blood pressure were observed and maintaining the patients in the recumbent position was sufficient to alleviate symptoms. In severe cases use of the Trendelenburg position should be enough to correct any alterations.

PIP: The effects of the insertion of an intrauterine device (IUD), endometrial biopsy, and uterine flushing on electrocardiograph patterns were evaluated in a prospective study of 260 women. The frequency of bradycardia was significantly higher during insertion of a Lippes loop IUD (p less than .001) or Cu-7 IUD (p less than .02) than with other procedures. The frequency of tachycardia was greater than that of bradycardia during the insertion of an uterine progesteronr system and TCu-200 IUD. However, the severity of bradycardia was similar in all groups. The frequency of bradycardia was similar among nulliparous women, and was correlated with reports of pain during any of the procedures. Changes in blood pressure were not severe, and symptoms were relieved by maintaining patients in a recumbent position. The Trendelenburg position is recommended in severe cases.