The Effects of Adding Particles in Texture Modified Food on Tongue Strength and Swallowing Function in Patients with Oropharyngeal Dysphagia: A Proof of Concept Study.

The use of texture modified food (TMF) is widely spread in the daily care of patients with oropharyngeal dysphagia (OD). However, TMF have been shown to have a negative impact on the patients' quality of life. Adherence rates are low, increasing the risk of malnutrition and aspiration in an already vulnerable patient population. The aim of this exploratory study was to gain insight in the feasibility of adding particles to pureed food on tongue strength, swallowing safety and efficiency in patients with OD. Ten adult participants with OD swallowed three different boluses. Bolus 1 consisted of no particles (IDDSI level 4), small and bigger particles were added in bolus 2 and 3. Tongue strength during swallowing (Pswal) was measured using the Iowa Oral Performance Instrument (IOPI). Swallow safety (penetration and aspiration) and swallow efficiency (residu) were quantified during fiberoptic endoscopic evaluation of swallowing by means of the PAS scale and Pooling score. RM Anova and Friedman tests were performed for analyzing the impact of bolus on the outcome parameters. No significant effect of bolus type on Pswal was measured. Neither the PAS nor the Pooling score differed significantly between the three different boluses. Aspiration was never observed during swallowing any bolus with particles. This preliminary study shows that the addition of particles to pureed food had no impact on Pswal, swallowing efficiency or safety in patients with OD. This innovative project is the first step in research to explore the characteristics of TMF beyond bolus volume, viscosity and temperature.

Odor Identification by Parkinson's Disease Patients Tested by Using Sniffin' Sticks versus Natural Spices.

Introduction: Hyposmia is a frequent symptom of Parkinson's disease (PD), which greatly impacts patients' flavor perception and their quality of life. However, PD patients recognize some odors better than others. Identifying which food odors are still recognized by PD patients may be useful for flavor enhancement. Our aim was to evaluate the olfactory identification of Sniffin' Sticks and spice odorants in PD patients and healthy controls (HC), to identify the impact of synthetic odorants compared with real-life food and the impact of odor familiarity and pleasantness on odorant identification in PD patients. Methods: Sniffin' Sticks odorant identification was evaluated in 80 PD patients and 105 age-matched HC. In a subset, the spice odorant identification was evaluated. Results: The mean total score was higher for the Sniffin' Sticks than for the spice odor identification test in all participants (55.4% versus 22.5%). Sniffin' Sticks orange, peppermint, rose, and fish odorants were best correctly identified by PD patients, by 62.5, 53.8, 52.9, and 57.5%, respectively. Of the spice odor identification test, garlic and "no stimulus" were best correctly identified by PD patients, by, respectively, 38.2 and 67.6%. HC identified most Sniffin' Sticks odorants and spices better than PD patients. Odorant familiarity determined real-life food odorant identification. Conclusion: This study demonstrates that some food odorants, both the commercial Sniffin' Sticks as natural odorants, are still recognized by PD patients. Sniffin' Sticks were better recognized compared with real-life odorants, by both HC and PD patients. Odorant familiarity determined PD patients' odorant identification; therefore, familiar food odorants may have potential for a future flavor enhancement. Implications. This is the first study, to our knowledge, to evaluate real-life food odor identification in PD patients. Our results provide a first step towards patient-appropriate flavor enhancement strategies in PD.

Chefs in Future Integrated Healthcare - Current State and Innovation Needs: A First Overview of the NECTAR Project (aN Eu Curriculum for Chef gasTro-Engineering in Primary Food Care).

People in need of care, chronic or acute, often present problematic food intake and special nutritional needs. Integrated, person-centred and pro-active food and nutritional care delivery has been proven effective for people in health care. However, skills mismatches have been reported in different professions involved, which also applies to the role of chefs in healthcare. The EU funded project NECTAR aims at closing this gap by creating a new job profile, called Chef Gastro-Engineering (CGE). The current publication summarizes the status quo in hospitals and gives a perspective on the future role of chefs in integrated healthcare delivery.

Identification and Characterization of Human Observational Studies in Nutritional Epidemiology on Gut Microbiomics for Joint Data Analysis.

In any research field, data access and data integration are major challenges that even large, well-established consortia face. Although data sharing initiatives are increasing, joint data analyses on nutrition and microbiomics in health and disease are still scarce. We aimed to identify observational studies with data on nutrition and gut microbiome composition from the Intestinal Microbiomics (INTIMIC) Knowledge Platform following the findable, accessible, interoperable, and reusable (FAIR) principles. An adapted template from the European Nutritional Phenotype Assessment and Data Sharing Initiative (ENPADASI) consortium was used to collect microbiome-specific information and other related factors. In total, 23 studies (17 longitudinal and 6 cross-sectional) were identified from Italy (7), Germany (6), Netherlands (3), Spain (2), Belgium (1), and France (1) or multiple countries (3). Of these, 21 studies collected information on both dietary intake (24 h dietary recall, food frequency questionnaire (FFQ), or Food Records) and gut microbiome. All studies collected stool samples. The most often used sequencing platform was Illumina MiSeq, and the preferred hypervariable regions of the 16S rRNA gene were V3-V4 or V4. The combination of datasets will allow for sufficiently powered investigations to increase the knowledge and understanding of the relationship between food and gut microbiome in health and disease.

Parkinson's disease patients' short chain fatty acids production capacity after in vitro fecal fiber fermentation.

Animal models indicate that butyrate might reduce motor symptoms in Parkinson's disease. Some dietary fibers are butyrogenic, but in Parkinson's disease patients their butyrate stimulating capacity is unknown. Therefore, we investigated different fiber supplements' effects on short-chain fatty acid production, along with potential underlying mechanisms, in Parkinson's patients and age-matched healthy controls. Finally, it was investigated if this butyrate production could be confirmed by using fiber-rich vegetables. Different fibers (n = 40) were evaluated by in vitro fermentation experiments with fecal samples of Parkinson's patients (n = 24) and age-matched healthy volunteers (n = 39). Short-chain fatty acid production was analyzed by headspace solid-phase micro-extraction gas chromatography-mass spectrometry. Clostridium coccoides and C. leptum were quantified through 16S-rRNA gene-targeted group-specific qPCR. Factors influencing short-chain fatty acid production were investigated using linear mixed models. After fiber fermentation, butyrate concentration varied between 25.6 ± 16.5 µmol/g and 203.8 ± 91.9 µmol/g for Parkinson's patients and between 52.7 ± 13.0 µmol/g and 229.5 ± 42.8 µmol/g for controls. Inulin had the largest effect, while xanthan gum had the lowest production. Similar to fiber supplements, inulin-rich vegetables, but also fungal ß-glucans, stimulated butyrate production most of all vegetable fibers. Parkinson's disease diagnosis limited short-chain fatty acid production and was negatively associated with butyrate producers. Butyrate kinetics during 48 h fermentation demonstrated a time lag effect in Parkinson's patients, especially in fructo-oligosaccharide fermentation. Butyrate production can be stimulated in Parkinson's patients, however, remains reduced compared to healthy controls. This is a first step in investigating dietary fiber's potential to increase short-chain fatty acids in Parkinson's disease.

Semi-industrial development of nutritious and healthy seafood dishes from sustainable species.

This study aimed to devise innovative, tailor-made, appealing, tasty and semi-industrialized dishes, using sustainable and under-utilized seafood species (bib, common dab, common carp, blue mussel and blue whiting), that can meet the specific nutritional and functional needs of children (8-10-years), pregnant women (20-40-years) and seniors (≥60-years). Hence, contests were organised among cooking schools from 6 European countries and the best recipes/dishes were reformulated, semi-industrially produced and chemically and microbiologically evaluated. The dishes intended for: (i) children and pregnant women had EPA + DHA and I levels that reached the target quantities, supporting the claim as "high in I"; and (ii) seniors were "high in protein" (24.8%-Soup_S and 34.0%-Balls_S of the energy was provided by proteins), "high in vitamin B12", and had Na contents (≤0.4%) below the defined limit. All dishes reached the vitamin D target value. Sausages_C, Roulade_P, Fillet_P and Balls_S had a well-balanced protein/fat ratio. Roulade_P presented the highest n-3 PUFA/n-6 PUFA ratio (3.3), while Sausages_C the lowest SFA/UNS ratio (0.2). Dishes were considered safe based on different parameters (e.g. Hg-T, PBDEs, Escherichia coli). All represent dietary sources contributing to meet the reference intakes of target nutrients (33->100%), providing valuable options to overcome nutritional and functional imbalances of the three groups.

Dysphagia management in Parkinson's disease: Comparison of the effect of thickening agents on taste, aroma, and texture.

Dysphagia is a frequent symptom in Parkinson's disease (PD). Thickening liquids facilitates safe swallowing, however, low treatment compliance is a major issue, due to patients' dislike of thickened liquids. Some studies suggest a negative impact of gum-based thickeners, currently most used in clinical practice, on sensory properties compared to starch-based thickeners. This has not yet been investigated in PD. This study's aim was to compare taste, texture, and aroma of gum-based and starch-based thickened soups in participants with PD. Gum-based resource thicken up clear (RTUC) and starch-based kitchen products potato starch (PS) and quinoa flour (QF) were evaluated in broccoli soup. Texture, aroma, and taste were characterized by rheology, volatile, and sensory profiling. Thickened soups were evaluated in participants with PD and controls through a paired comparison test. Reduced release of 61.4%, 46.2%, and 38.5% of volatiles was observed after thickening with RTUC, PS, and QF, respectively. Overall taste intensity was reduced in RTUC- and PS-thickened soup, respectively. Taste and aroma of PS-thickened soup were considered more intense by 70.3% and 63.8% of all participants, respectively (n = 36 PD, n = 41 controls), 56.3% preferred the PS-thickened soup's texture . Taste and aroma of QF-thickened soup were considered more intense by 68.1% and 65.6% of all participants, respectively (n = 47 PD, n = 31 controls), 58.0% preferred the QF-thickened soup's texture. Starch-based thickeners demonstrated higher taste and aroma intensity. However, volatile and sensory profiling demonstrated reduced taste and aroma in all thickeners. Combining kitchen products with flavor enhancers may increase palatability of thickened beverages.

Dietary Intake of Parkinson's Disease Patients.

Background and Aims: Dietary management, as an adjuvant therapy in Parkinson's disease (PD), provides clear benefits to patients. However, baseline information about the usual dietary intake of Parkinson's patients is lacking. Methods: We conducted an observational cross-sectional study, investigating the dietary intake in Belgian PD patients, as well as their medication use and knowledge of possible food-drug interactions. A dietary record of 2 non-consecutive days, allowing the calculation of usual intake, was used. Medication use and knowledge of food-drug interactions were investigated using a self-administered questionnaire. Results: The nutrient (both macro and micro) intake in this study was similar to the dietary pattern of the general Belgian population. However, results showed that the PD population had a high dietary fiber intake of 26.2 ± 7.7 g/day, which is in line with the recommended intake. The majority of the PD patients had an inadequate intake of vitamin D and iron (respectively, 55.9 and 76.5% of all participants). When looking into the knowledge about food-drug interactions, the majority of the PD patients claimed to be aware of the food-drug interaction between dietary proteins and levodopa. However, only 18.2% of the patients took all doses of levodopa out of meals. Conclusion: Our results show that monitoring of dietary intake in PD patients is of importance to detect possible micronutrient insufficiencies. Patients should receive professional guidance in optimizing their diet to accommodate for different complaints inherent to PD, including constipation. Furthermore, the knowledge of patients regarding the importance of correct medication intake should be improved.

First report of an idiopathic partial tail myonecrosis in European brown shrimp Crangon crangon caught in the North Sea.

The European brown shrimp Crangon crangon is an abundant and commercially important species in the North Sea. Currently, there is interest in landing live shrimp to provide fresh animals for a growing market in live brown shrimp. During 4 survival studies between 2014 and 2016, shrimp were collected from commercial trawlers and maintained alive in off-shore facilities. From Day 1 onwards, a minority of shrimp (~2.24%) developed a white discoloration of the abdominal muscles and a depigmentation of the distal part of the abdomen, along with paralysis of the affected tissues and appendages. As the symptoms progressed, a circumferential blackish delineation appeared, creating a distinct boundary between healthy and necrotic tissue. Affected shrimp survived up to 3 wk, although in several animals the distal part of the tail was completely lost. Histological and electron microscopical examination confirmed the myonecrosis. A secondary bacterial invasion of the necrotic muscle was observed in some animals. RT-PCR for infectious myonecrosis virus was negative. The condition appears not to be contagious, based on the feeding of healthy shrimp with necrotic tissue of affected shrimp. Based on these observations, a mechanical cause inflicted during the catching process is proposed.

Effects of ascorbic acid on patulin in aqueous solution and in cloudy apple juice.

Degradation of the mycotoxin patulin (PAT) and the generation of (less toxic) breakdown products, such as (E/Z)-ascladiol (ASC-E/Z) and desoxypatulinic acid (D-PAT), can occur due to chemical, physical and biological treatments. Our study focused on the chemical degradation of PAT in the presence of ascorbic acid (AA) both for pure PAT standard in acidified aqueous solution and for PAT-contaminated cloudy apple juice (CAJ) (obtained via addition of apple mash produced from apples inoculated with Penicillium expansum). Within this framework, different concentrations of AA were evaluated, as well as the presence/absence of oxygen and different storage temperatures. In order to do so, an in-house methodology allowing a good separation of PAT from its reaction and breakdown products was optimized first. The highest PAT reduction (60%) in CAJ with an initial PAT concentration of 100 µg/kg and 0.25% (w/v) AA was achieved after 6 days of incubation at 22 °C in the presence of oxygen. It was also found that the treatment by AA resulted in the generation of degradation products less toxic than PAT (such as (E/Z)-ASC). In conclusion, AA used to improve numerous product quality aspects (e.g. colour (less browning), nutritional value, etc.) and considered as a safe food additive (Food and Drug Administration (FDA) (1999)) has an effect on PAT degradation. It was shown that such degradation generated less toxic compounds in the presence of oxygen. In view of consumers' safety, fortification of apple juice (and possibly apple-based products) with AA could be helpful within an integrated system to ensure products with low levels of patulin. The optimum conditions for such an approach within a legal and practical point of view need to be further explored.

Kinetics of drosopterin release as indicator pigment for heat-induced color changes of brown shrimp (Crangon crangon).

Heat-induced color changes of crustaceans are commonly described as the release of astaxanthin. In this study on Crangon crangon, it was found that astaxanthin plays a minor role in the (dis)coloration. By LC-HRMS, two polar, process dependent pigments were found. One pigment was identified as riboflavin and one as drosopterin (level-2 certainty). Thermal treatments had highest effect on drosopterin concentration changes and were chosen as indicator for a kinetic study of heat-induced color changes. The kinetic data fitted a consecutive step model (r2 = 0.971), including a first step in which drosopterin was released (kd,85°C = 0.95 ±â€¯0.09 min-1; Ead = 105 ±â€¯4 kJ/mol) and a second step where drosopterin is degraded (kb,85°C = 0.02 ±â€¯0.002 min-1; Eab = 190 ±â€¯15 kJ/mol). The kinetic model shows that shrimp should be heated at lower temperatures (<80 °C) than the heating temperatures used by fishermen (86-101 °C), creating opportunities for quality optimization. Therefore, this study delivers essential information needed in a comprehensive quality optimization study of the cooked brown shrimp.

Thermal inactivation kinetics of proteases and polyphenoloxidase in brown shrimp (Crangon crangon).

To optimize product quality of the cooked brown shrimp (Crangon crangon), quantitative data on the influence of all relevant process parameters (treatment time and temperature) on several quality attributes is required. Surprisingly, kinetic data and models on heat induced inactivation of important endogenous spoilage enzymes of the brown shrimp are not available today. In this study the thermal inactivation kinetics of the most important spoilage enzymes, proteases and polyphenoloxidase (PPO), were determined from isothermal heat treatments of enzyme extracts of the cephalothorax. For both enzymes, inactivation kinetics showed first order decay(s). Proteases showed two distinct stability fractions. A labile fraction, representing 42±2% of the total activity with kl,60°C=0.94±0.14 min(-1) and Ea,l=178±8.5 kJ/mol, and a stable fraction, representing 58±2%, with ks,60°C=0.020±0.002 min(-1) and Ea,s=155±7.0 kJ/mol. PPO showed a single fraction with k60°C=1.58±0.02 min(-1) and Ea=161±2.2 kJ/mol. Based on these results, the proteolytic activity, in particular the thermostable fraction, should be considered as a target in thermal processing of brown shrimp in relation to enzyme induced product quality changes during storage.

Detection and identification of xerophilic fungi in Belgian chocolate confectionery factories.

Chocolate confectionery fillings are generally regarded as microbiologically stable. The stability of these fillings is largely due to the general practice of adding either alcohol or preservatives. Consumer demands are now stimulating producers to move away from adding alcohol or other preservatives to their confectionery fillings and instead to search for innovative formulations. Such changes in composition can influence the shelf life of the product and may lead to spoilage by xerophilic fungi. The aim of this study was to test whether the production environment of Belgian chocolate confectionery factories and common ingredients of chocolate confectioneries could be potential sources of contamination with xerophilic fungal species. In the factory environment, the general and strictly xerophilic fungal spore load was determined using an RCS Air Sampler device in combination with DG18 and MY50G medium, respectively. Four basic ingredients of chocolate confectionery fillings were also examined for fungal spore levels using a direct plating technique. Detected fungi were identified to species level by a combination of morphological characterization and sequence analysis. Results indicated a general fungal spore load in the range of 50-250 colony forming units per cubic meter of air (CFU/m(3) air) and a more strict xerophilic spore load below 50 CFU/m(3) air. These results indicate rather low levels of fungal spores present in the factory environment. The most prevalent fungi in the factory environment were identified as Penicillium spp., particularly Penicillium brevicompactum. Examination of the basic ingredients of confectionery fillings revealed nuts to be the most likely potential source of direct contamination. In nuts, the most prevalent fungal species identified were Eurotium, particularly Eurotium repens.

Enteric pathogen survival varies substantially in irrigation water from Belgian lettuce producers.

It is accepted that irrigation water is a potential carrier of enteric pathogens, such as Salmonella and E. coli O157:H7 and, therefore, a source for contamination of fresh produce. We tested this by comparing irrigation water samples taken from five different greenhouses in Belgium. The water samples were inoculated with four zoonotic strains, two Salmonella and two E. coli O157:H7 strains, and pathogen survival and growth in the water were monitored up till 14 days. The influence of water temperature and chemical water quality was evaluated, and the survival tests were also performed in water samples from which the resident aquatic microbiota had previously been eliminated by filter sterilization. The pathogen's survival differed greatly in the different irrigation waters. Three water samples contained nutrients to support important growth of the pathogens, and another enabled weaker growth. However, for all, growth was only observed in the samples that did not contain the resident aquatic microbiota. In the original waters with their specific water biota, pathogen levels declined. The same survival tendencies existed in water of 4 °C and 20 °C, although always more expressed at 20 °C. Low water temperatures resulted in longer pathogen survival. Remarkably, the survival capacity of two E. coli 0157:H7 strains differed, while Salmonella Thompson and Salmonella Typhimurium behaved similarly. The pathogens were also transferred to detached lettuce leaves, while suspended in two of the water samples or in a buffer. The effect of the water sample on the pathogen's fitness was also reproduced on the leaves when stored at 100% relative humidity. Inoculation of the suspension in buffer or in one of the water samples enabled epiphytic growth and survival, while the pathogen level in the other water sample decreased once loaded on the leaves. Our results show that irrigation waters from different origin may have a different capacity to transmit enteric pathogens and an important impact on the fitness of the pathogens to sustain and even grow on the leaf surface.

Evaluation of an attachment assay on lettuce leaves with temperature- and starvation-stressed Escherichia coli O157:H7 MB3885.

Attachment of enteric pathogens such as Escherichia coli O157:H7 to fresh produce is a crucial first step for contamination to occur, and irrigation water (IW) is considered a potentially important preharvest introduction route. In a natural situation, E. coli O157:H7 may be present in the irrigation water for some time and may, therefore, be starved. Most research, however, is performed with freshly cultured strains. The aim of this study was to examine the behavior of E. coli O157:H7 MB3885 under starvation stress in water used for overhead irrigation in the greenhouse and the consequence on its subsequent ability to attach to butterhead lettuce leaves. E. coli O157:H7 MB3885 was starvation stressed by introducing it at ±7.5 log CFU/ml into phosphate-buffered saline (PBS), sterile distilled water (SDW), or IW. The suspensions were stored at 4 or 20°C and were used after 0, 2, and 6 days for an attachment assay on butterhead lettuce. E. coli O157:H7 MB3885 levels were determined by plating method and live and dead quantitative PCR technique. A decrease in plate counts, an indicator of stress, was observed for most of the conditions, whereas a die-off, as revealed by the live and dead quantitative PCR data, was only observed in IW stored at 20°C. Overall, stress appeared to be highest in IW and lowest in PBS. The stressed cells were still able to recover, even at 4 °C, and to attach to the lettuce. Furthermore, our results show that standard laboratory solutions such as PBS and SDW may not be the best to simulate stressed cells in IW, in which the bacteria may behave significantly differently.

Volatile compounds associated with Psychrobacter spp. and Pseudoalteromonas spp., the dominant microbiota of brown shrimp (Crangon crangon) during aerobic storage.

The spoilage potential of several Psychrobacter and Pseudoalteromonas species (Psychrobacter cibarius, Psychrobacter maritimus, Pseudoalteromonas elyakovii, Pseudoalteromonas paragorgicola and Pseudoalteromonas nigrifaciens) was determined and quantified based on the presence of volatile organic compounds (VOCs). Psychrobacter and Pseudoalteromonas species dominate the microbiota of cooked brown shrimp (Crangon crangon). Additionally, API ZYM analyses determined the species' enzymatic capacity to contribute to spoilage by degrading lipids, amino acids and proteins. The bacterial species used in this study were isolated from cooked brown shrimp during storage under different storage and processing conditions and were selected for analysis of their spoilage potential based on their difference in the (GTG)5-rep profile, 16S rRNA and gyrB sequences and API ZYM profile. The isolates were inoculated as pure cultures on heat-sterilised shrimp. The inoculated samples were stored at 4 °C and the production of VOCs by the pure strains on the shrimp matrix was identified via gas chromatography coupled to mass spectrometry (GC-MS). VOC production was quantified daily by selected ion flow tube mass spectrometry (SIFT-MS) until the bacterial count exceeded 108-109 cfu/g. The sensory profile of Psychrobacter species revealed very low spoilage potential as measured by the production of VOCs, but these species may nevertheless contribute to spoilage. Based on the API ZYM results, Pseudoalteromonas as well as Psychrobacter species might enhance spoilage by breaking down lipids and hydrolysing amino acids and proteins. Pseudoalteromonas species, especially Psa. elyakovii and Psa. nigrifaciens, have a high spoilage potential and might be responsible for the off-odours produced during spoilage of brown shrimp. These isolates produced significant amounts of volatile compounds such as sulphides, acetone, ammonia, and ethanol, which are all involved in seafood spoilage.

Molecular identification of the microbiota of peeled and unpeeled brown shrimp (Crangon crangon) during storage on ice and at 7.5 °C.

The dominant microbiota of brown shrimp (Crangon crangon) were systematically identified during storage under different conditions. Freshly caught shrimp were processed on board the fishing vessel under the best possible hygienic conditions (IDEAL), unpeeled and manually (sterile) peeled, then stored on ice and at 7.5 °C until microbiologically spoiled. Results were compared with industrially processed (INDUSTRIAL) shrimp. Isolates grown on various media were identified by 16S rRNA and gyrB gene sequencing. We examined the total microbiota and microbial population shifts of shrimp under various storage conditions using denaturant gradient gel electrophoresis (DGGE). The microbiota differed somewhat during storage and among the various storage conditions; however, members of the genera Psychrobacter and Pseudoalteromonas were found to dominate the microbiota of all shrimp samples regardless of processing procedures or storage conditions. Most isolates could be identified by gyrB gene sequencing as Psychrobacter immobilis or Psychrobacter cibarius. Also Pseudoalteromonas nigrifaciens, Pseudoalteromonas elyakovii or Pseudoalteromonas paragorgicola dominated the microbiota of brown shrimp during storage. Also species from the genera Planocuccus, Exiguobacterium, Carnobacterium, Pseudomonas, Chryseobacterium and Staphylococcus were detected during storage of brown shrimp. Culture-dependent and culture-independent DGGE analysis produced different results in band patterns. Both methods are therefore required to accurately identify the microbiota and bacterial population shifts on seafood during storage.

Survival of enteric pathogens during butterhead lettuce growth: crop stage, leaf age, and irrigation.

The survival of Salmonella enterica serovar Thompson and Escherichia coli O157 was investigated on growing butterhead lettuce plants in the plant-growth chamber and greenhouse. All inoculation tests were made under conditions that approximate the greenhouse conditions for butterhead lettuce cultivation in Flanders (Belgium). The survival and proliferation of the pathogens on the leaves was determined at days 0, 4, and 8 after inoculation using standard plating techniques on selective medium. In the growth chamber, the extent to which both pathogens were able to multiply on the lettuce leaves was influenced by crop stage and leaf age. On young plants, the older leaves supported pathogen survival better. On nearly mature plants, pathogen population sizes were significantly higher on the old and young leaves compared with middle-aged leaves (p<0.001). In the greenhouse, the environmental regimen with high fluctuations in temperature and relative humidity was less conducive to the survival of E. coli O157, though its survival on nearly mature lettuce was enhanced by overhead irrigation. The moist conditions between the folded inner leaves are likely contributing to the survival of enteric pathogens in the lettuce head. Butterhead lettuce grown in greenhouses with a sprinkle irrigation system may present a potential health hazard when contaminated near harvest. Experimental design (growth chamber versus greenhouse) largely influences enteric pathogen behavior on growing lettuce plants.

Long-term survival of Escherichia coli O157:H7 and Salmonella enterica on butterhead lettuce seeds, and their subsequent survival and growth on the seedlings.

The long-term survival of enteric pathogens on butterhead lettuce seeds, and their subsequent survival and growth on seedlings were investigated. Lettuce seeds were inoculated at a high level with two Salmonella enterica and two Escherichia coli O157:H7 strains each (±8 log10 CFU/g seed) and the survival of the pathogens was monitored over two years using standard plating techniques on selective medium. The Salmonella strains (serovars Typhimurium and Thompson) survived significantly better on the seeds than the E. coli O157:H7 strains (MB3885 and NCTC12900). When individual seeds were tested two years after inoculation, Salmonella was recovered from each individual seed, whereas E. coli O157:H7 only from 4% to 14% of the seeds, depending on the recovery method. When contaminated stored seeds were germinated and the seedlings examined for presence of the pathogens, it was clear that both pathogens were able to proliferate on the seedlings. Pathogen counts up to 5.92 log10 CFU and 4.41 log10 CFU per positive seedling were observed for Salmonella and E. coli O157:H7, respectively. Our study not only confirms the long-term survival of enteric pathogens on seeds but also shows that the pathogens maintain their ability to resuscitate and proliferate on the seedlings. Seeds or seedlings should be considered as contamination sources for the cultivation of leafy vegetables such as butterhead lettuce grown in greenhouses.

Restriction analysis of an amplified rodA gene fragment to distinguish Aspergillus fumigatus var. ellipticus from Aspergillus fumigatus var. fumigatus.

A previous multidisciplinary study indicated that gliotoxin-producing Aspergillus fumigatus Fresen. isolates from silage commodities mostly belonged to its variant A. fumigatus var. ellipticus Raper & Fennell. Sequence analysis revealed the presence of a single nucleotide polymorphism at five positions in a fragment of the rodA gene (coding for a hydrophobin rodletA protein) between Aspergillus fumigatus var. fumigatus and Aspergillus fumigatus var. ellipticus. A method was developed to distinguish these two types of isolates based on restriction analysis of this rodA gene fragment using the HinfI restriction enzyme. In addition, in silico analysis of 113 rodA gene fragments retrieved from GenBank was performed and confirmed the suitability of this method. In conclusion, the method developed in this study allows easy distinction between A. fumigatus var. fumigatus and its variant ellipticus. In combination with the earlier developed PCR-restriction fragment length polymorphism method of Staab et al. (2009, J Clin Microbiol 47: 2079), this method is part of a sequencing-independent identification scheme that allows for rapid distinction between similar species/variants within Aspergillus section Fumigati, specifically A. fumigatus, A. fumigatus var. ellipticus, Aspergillus lentulus Balajee & K.A. Marr, Neosartorya pseudofischeri S.W. Peterson and Neosartorya udagawae Y. Horie, Miyaji & Nishim.