Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains.

Brettanomyces (Dekkera) bruxellensis is an ascomycetous yeast of major importance in the food, beverage and biofuel industry. It has been isolated from various man-made ecological niches that are typically characterized by harsh environmental conditions such as wine, beer, soft drink, etc. Recent comparative genomics studies revealed an immense intraspecific diversity, but it is still unclear whether this genetic diversity also leads to systematic differences in fermentation performance and (off-)flavor production, and to what extent strains have evolved to match their ecological niche. Here, we present an evaluation of the fermentation properties of eight genetically diverse B. bruxellensis strains originating from beer, wine and soft drinks. We show that sugar consumption and aroma production during fermentation are determined by both the yeast strain and composition of the medium. Furthermore, our results indicate a strong niche adaptation of B. bruxellensis, most clearly for wine strains. For example, only strains originally isolated from wine were able to thrive well and produce the typical Brettanomyces-related phenolic off-flavors 4-ethylguaiacol and 4-ethylphenol when inoculated in red wine. Sulfite tolerance was found as a key factor explaining the observed differences in fermentation performance and off-flavor production. Sequence analysis of genes related to phenolic off-flavor production, however, revealed only marginal differences between the isolates tested, especially at the amino acid level. Altogether, our study provides novel insights in the Brettanomyces metabolism of flavor production, and is highly relevant for both the wine and beer industry.

Phylogenetic signal in phenotypic traits related to carbon source assimilation and chemical sensitivity in Acinetobacter species.

A common belief is that the phylogeny of bacteria may reflect molecular functions and phenotypic characteristics, pointing towards phylogenetic conservatism of traits. Here, we tested this hypothesis for a large set of Acinetobacter strains. Members of the genus Acinetobacter are widespread in nature, demonstrate a high metabolic diversity and are resistant to several environmental stressors. Notably, some species are known to cause opportunistic human infections. A total of 133 strains belonging to 33 species with validly published names, two genomic species and species of an as-yet unknown taxonomic status were analyzed using the GENIII technology of Biolog, which allows high-throughput phenotyping. We estimated the strength and significance of the phylogenetic signal of each trait across phylogenetic reconstructions based on partial RNA polymerase subunit B (rpoB) and core genome sequences. Secondly, we tested whether phylogenetic distance was a good predictor of trait differentiation by Mantel test analysis. And finally, evolutionary model fitting was used to determine if the data for each phenotypic character was consistent with a phylogenetic or an essentially random model of trait distribution. Our data revealed that some key phenotypic traits related to substrate assimilation and chemical sensitivity are linked to the phylogenetic placement of Acinetobacter species. The strongest phylogenetic signals found were for utilization of different carbon sources such as some organic acids, amino acids and sugars, thus suggesting that in the diversification of Acinetobacter carbon source assimilation has had a relevant role. Future work should be aimed to clarify how such traits have shaped the remarkable ability of this bacterial group to dominate in a wide variety of habitats.

Microbiology of sugar-rich environments: diversity, ecology and system constraints.

Microbial habitats that contain an excess of carbohydrate in the form of sugar are widespread in the microbial biosphere. Depending on the type of sugar, prevailing water activity and other substances present, sugar-rich environments can be highly dynamic or relatively stable, osmotically stressful, and/or destabilizing for macromolecular systems, and can thereby strongly impact the microbial ecology. Here, we review the microbiology of different high-sugar habitats, including their microbial diversity and physicochemical parameters, which act to impact microbial community assembly and constrain the ecosystem. Saturated sugar beet juice and floral nectar are used as case studies to explore the differences between the microbial ecologies of low and higher water-activity habitats respectively. Nectar is a paradigm of an open, dynamic and biodiverse habitat populated by many microbial taxa, often yeasts and bacteria such as, amongst many others, Metschnikowia spp. and Acinetobacter spp., respectively. By contrast, thick juice is a relatively stable, species-poor habitat and is typically dominated by a single, xerotolerant bacterium (Tetragenococcus halophilus). A number of high-sugar habitats contain chaotropic solutes (e.g. ethyl acetate, phenols, ethanol, fructose and glycerol) and hydrophobic stressors (e.g. ethyl octanoate, hexane, octanol and isoamyl acetate), all of which can induce chaotropicity-mediated stresses that inhibit or prevent multiplication of microbes. Additionally, temperature, pH, nutrition, microbial dispersion and habitat history can determine or constrain the microbiology of high-sugar milieux. Findings are discussed in relation to a number of unanswered scientific questions.

Assessing the potential of wild yeasts for bioethanol production.

Bioethanol fermentations expose yeasts to a new, complex and challenging fermentation medium with specific inhibitors and sugar mixtures depending on the type of carbon source. It is, therefore, suggested that the natural diversity of yeasts should be further exploited in order to find yeasts with good ethanol yield in stressed fermentation media. In this study, we screened more than 50 yeast isolates of which we selected five isolates with promising features. The species Candida bombi, Wickerhamomyces anomalus and Torulaspora delbrueckii showed better osmo- and hydroxymethylfurfural tolerance than Saccharomyces cerevisiae. However, S. cerevisiae isolates had the highest ethanol yield in fermentation experiments mimicking high gravity fermentations (25 % glucose) and artificial lignocellulose hydrolysates (with a myriad of inhibitors). Interestingly, among two tested S. cerevisiae strains, a wild strain isolated from an oak tree performed better than Ethanol Red, a S. cerevisiae strain which is currently commonly used in industrial bioethanol fermentations. Additionally, a W. anomalus strain isolated from sugar beet thick juice was found to have a comparable ethanol yield, but needed longer fermentation time. Other non-Saccharomyces yeasts yielded lower ethanol amounts.

Assessing genetic diversity among Brettanomyces yeasts by DNA fingerprinting and whole-genome sequencing.

Brettanomyces yeasts, with the species Brettanomyces (Dekkera) bruxellensis being the most important one, are generally reported to be spoilage yeasts in the beer and wine industry due to the production of phenolic off flavors. However, B. bruxellensis is also known to be a beneficial contributor in certain fermentation processes, such as the production of certain specialty beers. Nevertheless, despite its economic importance, Brettanomyces yeasts remain poorly understood at the genetic and genomic levels. In this study, the genetic relationship between more than 50 Brettanomyces strains from all presently known species and from several sources was studied using a combination of DNA fingerprinting techniques. This revealed an intriguing correlation between the B. bruxellensis fingerprints and the respective isolation source. To further explore this relationship, we sequenced a (beneficial) beer isolate of B. bruxellensis (VIB X9085; ST05.12/22) and compared its genome sequence with the genome sequences of two wine spoilage strains (AWRI 1499 and CBS 2499). ST05.12/22 was found to be substantially different from both wine strains, especially at the level of single nucleotide polymorphisms (SNPs). In addition, there were major differences in the genome structures between the strains investigated, including the presence of large duplications and deletions. Gene content analysis revealed the presence of 20 genes which were present in both wine strains but absent in the beer strain, including many genes involved in carbon and nitrogen metabolism, and vice versa, no genes that were missing in both AWRI 1499 and CBS 2499 were found in ST05.12/22. Together, this study provides tools to discriminate Brettanomyces strains and provides a first glimpse at the genetic diversity and genome plasticity of B. bruxellensis.

Phenotypic evaluation of natural and industrial Saccharomyces yeasts for different traits desirable in industrial bioethanol production.

Saccharomyces cerevisiae is the organism of choice for many food and beverage fermentations because it thrives in high-sugar and high-ethanol conditions. However, the conditions encountered in bioethanol fermentation pose specific challenges, including extremely high sugar and ethanol concentrations, high temperature, and the presence of specific toxic compounds. It is generally considered that exploring the natural biodiversity of Saccharomyces strains may be an interesting route to find superior bioethanol strains and may also improve our understanding of the challenges faced by yeast cells during bioethanol fermentation. In this study, we phenotypically evaluated a large collection of diverse Saccharomyces strains on six selective traits relevant for bioethanol production with increasing stress intensity. Our results demonstrate a remarkably large phenotypic diversity among different Saccharomyces species and among S. cerevisiae strains from different origins. Currently applied bioethanol strains showed a high tolerance to many of these relevant traits, but several other natural and industrial S. cerevisiae strains outcompeted the bioethanol strains for specific traits. These multitolerant strains performed well in fermentation experiments mimicking industrial bioethanol production. Together, our results illustrate the potential of phenotyping the natural biodiversity of yeasts to find superior industrial strains that may be used in bioethanol production or can be used as a basis for further strain improvement through genetic engineering, experimental evolution, or breeding. Additionally, our study provides a basis for new insights into the relationships between tolerance to different stressors.

Assessing the xylanolytic bacterial diversity during the malting process.

The presence of microorganisms producing cell wall hydrolyzing enzymes such as xylanases during malting can improve mash filtration behavior and consequently have potential for more efficient wort production. In this study, the xylanolytic bacterial community during malting was assessed by isolation and cultivation on growth media containing arabinoxylan, and identification by 16S rRNA gene sequencing. A total of 33 species-level operational taxonomic units (OTUs) were found, taking into account a 3% sequence dissimilarity cut-off, belonging to four phyla (Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria) and 25 genera. Predominant OTUs represented xylanolytic bacteria identified as Sphingobacterium multivorum, Stenotrophomonas maltophilia, Aeromonas hydrophila and Pseudomonas fulva. DNA fingerprinting of all xylanolytic isolates belonging to S. multivorum obtained in this study revealed shifts in S. multivorum populations during the process. Xylanase activity was determined for a selection of isolates, with Cellulomonas flavigena showing the highest activity. The xylanase of this species was isolated and purified 23.2-fold by ultrafiltration, 40% ammonium sulfate precipitation and DEAE-FF ion-exchange chromatography and appeared relatively thermostable. This study will enhance our understanding of the role of microorganisms in the barley germination process. In addition, this study may provide a basis for microflora management during malting.

Characterization of the bacterial community composition in water of drinking water production and distribution systems in Flanders, Belgium.

The quality of drinking water is influenced by its chemical and microbial composition which in turn may be affected by the source water and the different processes applied in drinking water purification systems. In this study, we investigated the bacterial diversity in different water samples from the production and distribution chain of thirteen drinking water production and distribution systems from Flanders (Belgium) that use surface water or groundwater as source water. Water samples were collected over two seasons from the source water, the processed drinking water within the production facility and out of the tap in houses along its distribution network. 454-pyrosequencing of 16S ribosomal RNA gene sequences revealed a total of 1,570 species-level bacterial operational taxonomic units. Strong differences in community composition were found between processed drinking water samples originating from companies that use surface water and other that use groundwater as source water. Proteobacteria was the most abundant phylum in all samples. Yet, several phyla including Actinobacteria were significantly more abundant in surface water while Cyanobacteria were more abundant in surface water and processed water originating from surface water. Gallionella, Acinetobacter, and Pseudomonas were the three most abundant genera detected. Members of the Acinetobacter genus were even found at a relative read abundance of up to 47.5% in processed water samples, indicating a general occurrence of Acinetobacter in drinking water (systems).

Present knowledge of the bacterial microflora in the extreme environment of sugar thick juice.

The diversity of the bacterial population in sugar thick juice, an intermediate product in the production of beet sugar, which exhibits an extreme, osmophilic environment with a water activity value (a(w)) less than 0.86, was assessed with both culture-dependent and -independent 16S ribosomal RNA (rRNA) gene-based analyses. In comparison with previous studies, the number of different thick juice bacterial species increased from 29 to 72. Remarkably, a limited, gram-positive, culturable flora, encompassing species of Bacillus, Staphylococcus and mainly Tetragenococcus dominated thick juice during storage, while a more heterogeneous and unculturable fraction of Acinetobacter, Sporolactobacillus and Thermus species could be detected in freshly produced thick juice. Notably, almost all bacteria detected in the thick juice were also detected in the air, emphasising the importance of further investigation and assessment of strategies to reduce (air) contamination during processing and storage. The discovery of the contamination source may be used for the development of management strategies for thick juice degradation resulting from microbial activity.

Predominance of Tetragenococcus halophilus as the cause of sugar thick juice degradation.

The industrial storage of sugar thick juice was simulated on a laboratory scale. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis and the application of Clone Libraries in parallel with classical microbiology were used to study the bacterial diversity and all revealed a dominance (>99%) of Tetragenococcus halophilus during storage. The degradation of thick juice correlated with the appearance of L-lactic acid and high concentrations of T. halophilus. In addition, pure cultures of T. halophilus induced degradation of sterile thick juice. A specific PCR was developed to detect T. halophilus and industrial thick juice samples from Belgium, Germany and France all contained T. halophilus, suggesting a consistent association of this organism with thick juice. T. halophilus has been known only as a halophile thus far, and this report is the first to show an association of this organism with a sugar-rich environment.

Isolation and screening of bacterial isolates from wastewater treatment plants to decolorize azo dyes.

The discharge of dye-contaminated wastewater into natural waterways presents a substantial risk to human and environmental health, therefore necessitating the treatment and removal of toxic dyes from colored wastewaters before their release into the ecosystem. The aim of this study was to isolate and characterize bacterial strains capable of decolorizing and/or degrading azo dyes commonly applied in textile production (monoazo dye Reactive Orange 16 and diazo dye Reactive Green 19) from activated sludge systems used in the treatment of (textile) wastewater. Following a prescreening of 125 isolates for their decolorization potential five strains were retained for further evaluation of decolorization rate and effects of physicochemical parameters using a microtiter plate method. Of those five strains, one strain belonging to the genus Acinetobacter (ST16.16/164) and another belonging to Klebsiella (ST16.16/034) outperformed the other tested strains. Both strains exhibited strong decolorization ability (>80%) within a wide temperature range (20 °C-40 °C) and retained good decolorization activity at temperatures as low as 10 °C (especially strain ST16.16/034). Among the different pH values tested (pH 4, 7 and 10), highest dye removal for both strains occurred at pH 7, with decolorization efficiency remaining relatively high under alkaline conditions (pH 10), and neither isolates decolorization efficiency was negatively impacted by high salt or high dye concentration. Furthermore, both strains displayed the highest rate of decolorization and were able to completely (ST16.16/034) or partly (ST16.16/164) degrade the azo dyes. Altogether, our results support the use of these bacteria in the treatment of industrial wastewaters containing azo dyes.

Decolorization of reactive azo dyes using a sequential chemical and activated sludge treatment.

Textile wastewater contains high concentrations of organic substances derived from diverse dyes and auxiliary chemicals, some of which are non-biodegradable and/or toxic. Therefore, it is essential that textile wastewater is treated and that these substances are removed before being discharged into the environment. A combination of advanced oxidation processes (AOPs) to obtain partial dye degradation followed by a biological treatment has been suggested as a promising method for cost-effective decolorization of wastewater. The aim of this study was to develop and evaluate a combined method of partial Fenton's oxidation and biological treatment using activated sludge for decolorization of azo dyes, which represent an important group of recalcitrant, toxic textile dyes. Using Reactive Violet 5 (RV5) as a model dye, color removal was significantly higher when the combined Fenton treatment/activated sludge method was used, as opposed to separate application of these treatments. More specifically, pretreatment with Fenton's reagent removed 52.9, 83.9 and 91.3 % of color from a 500 mg l-1 RV5 aqueous solution within 60 min when H2O2 concentrations of 1.0, 1.5, and 2.0 mM were used, respectively. Subsequent biological treatment was found to significantly enhance the chemical treatment, with microbial decolorization removing 70.2 % of the remaining RV5 concentration, on average. Molecular analysis of the microbial community within the activated sludge revealed that exposure to RV5 shifted the community composition from diverse towards a highly-specialized community harboring taxa with azo dye degrading activity, including Trichosporon, Aspergillus and Clostridium species.

Assessing the composition of microbial communities in textile wastewater treatment plants in comparison with municipal wastewater treatment plants.

It is assumed that microbial communities involved in the biological treatment of different wastewaters having a different chemical composition harbor different microbial populations which are specifically adapted to the environmental stresses encountered in these systems. Yet, little is known about the composition of these microbial communities. Therefore, the aim of this study was to assess the microbial community composition over two seasons (winter and summer) in activated sludge from well-operating textile wastewater treatment plants (WWTPs) in comparison with municipal WWTPs, and to explain observed differences by environmental variables. 454-pyrosequencing generated 160 archaeal and 1645 bacterial species-level Operational Taxonomic Units (OTUs), with lower observed richness in activated sludge from textile WWTPs compared to municipal WWTPs. The bacterial phyla Planctomycetes, Chloroflexi, Chlorobi, and Acidobacteria were more abundant in activated sludge samples from textile WWTPs, together with archaeal members of Thaumarchaeota. Nonmetric multidimensional scaling analysis of the microbial communities showed that microbial communities from textile and municipal WWTPs were significantly different, with a seasonal effect on archaea. Nitrifying and denitrifying bacteria as well as phosphate-accumulation bacteria were more abundant in municipal WWTPs, while sulfate-reducing bacteria were almost only detected in textile WWTPs. Additionally, microbial communities from textile WWTPs were more dissimilar than those of municipal WWTPs, possibly due to a wider diversity in environmental stresses to which microbial communities in textile WWTPs are subjected to. High salinity, high organic loads, and a higher water temperature were important potential variables driving the microbial community composition in textile WWTPs. This study provides a general view on the composition of microbial communities in activated sludge of textile WWTPs, and may provide novel insights for identifying key players performing important functions in the purification of textile wastewaters.

Quantitative farm-to-fork risk assessment model for norovirus and hepatitis A virus in European leafy green vegetable and berry fruit supply chains.

Fresh produce that is contaminated with viruses may lead to infection and viral gastroenteritis or hepatitis when consumed raw. It is thus important to reduce virus numbers on these foods. Prevention of virus contamination in fresh produce production and processing may be more effective than treatment, as sufficient virus removal or inactivation by post-harvest treatment requires high doses that may adversely affect food quality. To date knowledge of the contribution of various potential contamination routes is lacking. A risk assessment model was developed for human norovirus, hepatitis A virus and human adenovirus in raspberry and salad vegetable supply chains to quantify contributions of potential contamination sources to the contamination of produce at retail. These models were used to estimate public health risks. Model parameterization was based on monitoring data from European supply chains and literature data. No human pathogenic viruses were found in the soft fruit supply chains; human adenovirus (hAdV) was detected, which was additionally monitored as an indicator of fecal pollution to assess the contribution of potential contamination points. Estimated risks per serving of lettuce based on the models were 3×10(-4) (6×10(-6)-5×10(-3)) for NoV infection and 3×10(-8) (7×10(-10)-3×10(-6)) for hepatitis A jaundice. The contribution to virus contamination of hand-contact was larger as compared with the contribution of irrigation, the conveyor belt or the water used for produce rinsing. In conclusion, viral contamination in the lettuce and soft fruit supply chains occurred and estimated health risks were generally low. Nevertheless, the 97.5% upper limit for the estimated NoV contamination of lettuce suggested that infection risks up to 50% per serving might occur. Our study suggests that attention to full compliance for hand hygiene will improve fresh produce safety related to virus risks most as compared to the other examined sources, given the monitoring results. This effect will be further aided by compliance with other hygiene and water quality regulations in production and processing facilities.

Comparing the influence of low power ultrasonic and microwave pre-treatments on the solubilisation and semi-continuous anaerobic digestion of waste activated sludge.

Anaerobic digestion is a well-known technique for the recovery of energy from waste sludge. Pre-treatment methods are useful tools to improve the biodegradability of the sludge and to enhance the digestion efficiency. In this study, an ultrasound (US) and a microwave (MW) pre-treatment were compared in a long-term digestion experiment, using 3 small pilot scale semi-continuous digesters (SRT=20 days). A specific energy of 96 kJ/kg sludge was applied, hence enabling to compare the effectiveness of both pre-treatment methods towards sludge solubilisation and biogas production enhancement. Total and volatile solids (TS and VS), COD, carbohydrates and proteins were monitored throughout the digestion experiment. It was seen that US was most effective in COD solubilisation. The average biogas increment was 20% for the microwave pre-treatment and 27% for the ultrasonic pre-treatment. However, this additional biogas production did not outweigh the energy consumed by the pre-treatment, leading to a negative energy balance.

Rosenbergiella australoborealis sp. nov., Rosenbergiella collisarenosi sp. nov. and Rosenbergiella epipactidis sp. nov., three novel bacterial species isolated from floral nectar.

The taxonomic status of nine strains of the family Enterobacteriaceae isolated from floral nectar of wild Belgian, French, South African and Spanish insect-pollinated plants was investigated following a polyphasic approach. Confirmation that these strains belonged to the genus Rosenbergiella was obtained from comparative analysis of partial sequences of the 16S rRNA gene and other core housekeeping genes (atpD [ATP synthase ß-chain], gyrB [DNA gyrase subunit B] and rpoB [RNA polymerase ß-subunit]), DNA-DNA reassociation data, determination of the DNA G+C content and phenotypic profiling. Two strains belonged to the recently described species Rosenbergiella nectarea, while the other seven strains represented three novel species within the genus Rosenbergiella. The names Rosenbergiella australoborealis sp. nov. (with strain CdVSA 20.1(T) [LMG 27954(T)=CECT 8500(T)] as the type strain), Rosenbergiella collisarenosi sp. nov. (with strain 8.8A(T) [LMG 27955(T)=CECT 8501(T)] as the type strain) and Rosenbergiella epipactidis sp. nov. (with strain 2.1A(T) [LMG 27956(T)=CECT 8502(T)] as the type strain) are proposed. Additionally, the description of the genus Rosenbergiella is updated on the basis of new phenotypic and molecular data.

Among-population variation in microbial community structure in the floral nectar of the bee-pollinated forest herb Pulmonaria officinalis L.

BACKGROUND: Microbial communities in floral nectar have been shown to be characterized by low levels of species diversity, yet little is known about among-plant population variation in microbial community composition. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the microbial community structure (yeasts and bacteria) in floral nectar of ten fragmented populations of the bee-pollinated forest herb Pulmonaria officinalis. We also explored possible relationships between plant population size and microbial diversity in nectar, and related microbial community composition to the distance separating plant populations. Culturable bacteria and yeasts occurring in the floral nectar of a total of 100 plant individuals were isolated and identified by partially sequencing the 16S rRNA gene and D1/D2 domains of the 26S rRNA gene, respectively. A total of 9 and 11 yeast and 28 and 39 bacterial OTUs was found, taking into account a 3% (OTU0.03) and 1% sequence dissimilarity cut-off (OTU0.01). OTU richness at the plant population level (i.e. the number of OTUs per population) was low for yeasts (mean: 1.7, range: 0-4 OTUs0.01/0.03 per population), whereas on average 6.9 (range: 2-13) OTUs0.03 and 7.9 (range 2-16) OTUs0.01 per population were found for bacteria. Both for yeasts and bacteria, OTU richness was not significantly related to plant population size. Similarity in community composition among populations was low (average Jaccard index: 0.14), and did not decline with increasing distance between populations. CONCLUSIONS/SIGNIFICANCE: We found low similarity in microbial community structure among populations, suggesting that the assembly of nectar microbiota is to a large extent context-dependent. Although the precise factors that affect variation in microbial community structure in floral nectar require further study, our results indicate that both local and regional processes may contribute to among-population variation in microbial community structure in nectar.

Does virulence assessment of Vibrio anguillarum using sea bass (Dicentrarchus labrax) larvae correspond with genotypic and phenotypic characterization?

BACKGROUND: Vibriosis is one of the most ubiquitous fish diseases caused by bacteria belonging to the genus Vibrio such as Vibrio (Listonella) anguillarum. Despite a lot of research efforts, the virulence factors and mechanism of V. anguillarum are still insufficiently known, in part because of the lack of standardized virulence assays. METHODOLOGY/PRINCIPAL FINDINGS: We investigated and compared the virulence of 15 V. anguillarum strains obtained from different hosts or non-host niches using a standardized gnotobiotic bioassay with European sea bass (Dicentrarchus labrax L.) larvae as model hosts. In addition, to assess potential relationships between virulence and genotypic and phenotypic characteristics, the strains were characterized by random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (rep-PCR) analyses, as well as by phenotypic analyses using Biolog's Phenotype MicroArray™ technology and some virulence factor assays. CONCLUSIONS/SIGNIFICANCE: Virulence testing revealed ten virulent and five avirulent strains. While some relation could be established between serotype, genotype and phenotype, no relation was found between virulence and genotypic or phenotypic characteristics, illustrating the complexity of V. anguillarum virulence. Moreover, the standardized gnotobiotic system used in this study has proven its strength as a model to assess and compare the virulence of different V. anguillarum strains in vivo. In this way, the bioassay contributes to the study of mechanisms underlying virulence in V. anguillarum.

Tracing enteric viruses in the European berry fruit supply chain.

In recent years, numerous foodborne outbreaks due to consumption of berry fruit contaminated by human enteric viruses have been reported. This European multinational study investigated possible contamination routes by monitoring the entire food chain for a panel of human and animal enteric viruses. A total of 785 samples were collected throughout the food production chain of four European countries (Czech Republic, Finland, Poland and Serbia) during two growing seasons. Samples were taken during the production phase, the processing phase, and at point-of-sale. Samples included irrigation water, animal faeces, food handlers' hand swabs, swabs from toilets on farms, from conveyor belts at processing plants, and of raspberries or strawberries at points-of-sale; all were subjected to virus analysis. The samples were analysed by real-time (reverse transcription, RT)-PCR, primarily for human adenoviruses (hAdV) to demonstrate that a route of contamination existed from infected persons to the food supply chain. The analyses also included testing for the presence of selected human (norovirus, NoV GI, NoV GII and hepatitis A virus, HAV), animal (porcine adenovirus, pAdV and bovine polyomavirus, bPyV) and zoonotic (hepatitis E virus, HEV) viruses. At berry production, hAdV was found in 9.5%, 5.8% and 9.1% of samples of irrigation water, food handlers' hands and toilets, respectively. At the processing plants, hAdV was detected in one (2.0%) swab from a food handler's hand. At point-of-sale, the prevalence of hAdV in fresh raspberries, frozen raspberries and fresh strawberries, was 0.7%, 3.2% and 2.0%, respectively. Of the human pathogenic viruses, NoV GII was detected in two (3.6%) water samples at berry production, but no HAV was detected in any of the samples. HEV-contaminated frozen raspberries were found once (2.6%). Animal faecal contamination was evidenced by positive pAdV and bPyV assay results. At berry production, one water sample contained both viruses, and at point-of-sale 5.7% and 1.3% of fresh and frozen berries tested positive for pAdV. At berry production hAdV was found both in irrigation water and on food handler's hands, which indicated that these may be important vehicles by which human pathogenic viruses enter the berry fruit chain. Moreover, both zoonotic and animal enteric viruses could be detected on the end products. This study gives insight into viral sources and transmission routes and emphasizes the necessity for thorough compliance with good agricultural and hygienic practice at the farms to help protect the public from viral infections.