Risk-benefit Assessment of Foods in Sweden - Developing a Working Procedure.

Background: Assessments of food-related health risks and benefits performed by national or international experts are still to a large extent separate processes. Diets, foods, and even single food ingredients, may potentially be associated with both beneficial and adverse health effects. Therefore in some cases it is important to consider both health risks and benefits, by assessing the balance between potential risks and benefits, ideally by expressing risk and benefit on the same scale. In Sweden risk-benefit assessments in the food sector are the responsibility of the Risk and Benefit Assessment Department at the National Food Agency (NFA), which brings together the scientific disciplines toxicology, nutrition and microbiology and related fields. In 2012 a project was initiated to develop a general procedure for risk-benefit assessments at the NFA. The results of this project, and an in-house working procedure, have been published in a governmental report [1] and presented at the Scientific conference “Shaping the Future of Food Safety Together” hosted by the European Food Safety Authority (EFSA) in Milan, Italy, in October 2015 (http://www.efsaexpo2015.eu/). The objectives of the project were to: • Summarize previous national and international experiences in the field of risk-benefit assessment • Develop a working procedure for practical use at the NFA • Test the procedure in a case study Principles of Risk-benefit Assessment From the overview and evaluation of the risk and benefit assessment literature it was concluded that no international consensus on the general principles or approaches for conducting risk-benefit assessment of foods and food components has so far been reached. The workflow suggested by EFSA [2] was used as a starting point for the development of the proposed NFA procedure for risk-benefit assessment. Tiered, stepwise approaches have been the preferred procedure in the majority of published risk-benefit assessments. In such an approach the nature of the question and the availability and type of data on for example food composition, exposure and health consequences determine at what tier the assessment can be stopped. The advantage of a stepwise methodology is that it is conceptually easy to use by the assessors and promotes transparency of the process. The NFA Procedure for Risk-benefit Assessment The developed risk-benefit assessment procedure contains three steps, from a qualitative assessment of risks and benefits separately to a quantitative assessment expressing risks and benefits on the same scale: 1) Initial assessment of risks and benefits separately 2) Enhanced assessment where different metrics for risks and benefits are weighted 3) Expressing risks and benefits on the same scale, for example mortality or DALYs (Disability Adjusted Life Years). Fig. 1. The relation between the three different steps in the NFA working procedure (1). Dialogue between the risk-benefit assessor and the risk-benefit manager is crucial after each step Case Study of the Risk-benefit Assessment Procedure The procedure was applied in a case study to assess the risk and benefits with a decreased content of nitrite and salt in processed meat, when done in combination with a decreased maximum storage temperature [3]. The potential health benefits for the general population, in terms of lower risk of exceeding ADI and lower risk of high blood pressure of decreased nitrite and salt intake was weighed against an increased risk of Clostridium botulinum and Listeria monocytogenes infections. The two first steps of the procedure could be applied, and it was concluded that the reduction of salt and nitrite levels would only have marginal effects on public health. Moreover, this reduction would not result in increased growth of microorganisms. The reduction of storage temperature from 8°C to 5°C would however result in a positive effect due to a reduction of growth of L. monocytogenes, but no effect on growth of C. botulinum. Future Activities at NFA Considering the complexity and the continuous development of the risk-benefit assessment area there is a need for national and international collaboration. At NFA we have initiated collaborative work with the Karolinska Institutet in Stockholm in order to increase the awareness of risk-benefit assessment in the academic environment, as well as to acquire valuable input from other experts. To promote cooperation on this subject in a Nordic setting we aim to host a workshop on risk-benefit assessment methods for Nordic countries during 2016. We are also open for collaboration in the risk-benefit assessment area within the EU. Conclusions The described working procedure is based on current best practices on how to perform risk-benefit assessments. The NFA working procedure share many features with for example the BEPRARIBEAN [4], BRAFO [5] and EFSA procedures [2]. Thus, our intention has been to incorporate experience and knowledge from previously published assessments and suggested procedures into a suitable in-house method. Our risk-benefit procedure is applied to relevant risk assessment questions at NFA. The case study regarding reduction of salt and nitrite content of processed meat helped us to identify critical points in the working procedure since it covered various aspects in microbiology, nutrition and toxicology. In our work and report we have identified several challenges associated with risk-benefit assessments. This emphasizes the need to develop a commonly accepted and feasible working procedure within the EU. The complete report can be downloaded free from: http://www.livsmedelsverket.se/globalassets/rapporter/2014/2014_livsmedelsverket_24_risk_benefit_assessment_procedures.pdf

Molecular detection of virulence factors among food and clinical Enterococcus faecalis strains in South Brazil

The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the β-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and β-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains.

Biofilm formation on polystyrene under different temperatures by antibiotic resistant Enterococcus faecalis and Enterococcus faecium isolated from food

The ability of antibiotic resistant E. faecalis and E. faecium isolated from food to form biofilm at different temperatures in the absence or presence of 0.75% glucose was evaluated. A synergistic effect on biofilm at 10 °C, 28 °C, 37 °C and 45 °C and glucose was observed for E. faecalis and E. faecium.

First description of Adenovirus, Enterovirus, Rotavirus and Torque teno virus in water samples collected from the Arroio Dilúvio, Porto Alegre, Brazil / Primeira descrição de Adenovírus, Enterovírus, Rotavírus e Torque teno vírus em amostras de água coletadas do Arroio Dilúvio, Porto Alegre, Brasil

Adenovirus (AdV), enterovirus (EV), genogroup A rotaviruses (GARV) and Torque teno virus (TTV) are non-enveloped viral agents excreted in feces and so may contaminate water bodies. In the present study, the molecular detection of these viruses was performed in samples of surface water collected from the Arroio Dilúvio, a waterstream that crosses the city of Porto Alegre, RS, Brazil, receiving great volumes of non-treated sewage from a large urban area. Sampling was performed during 2009, in three different occasions (January, April and September). The highest detection rate was observed for EV (64.28%), followed by TTV (28.57%) and AdV (21.43%). Rotaviruses were not detected. More than on kind of tested virus was detected in five (35. 71%) of 14 samples. January was the month with the highest viral detection rate, being all samples, collected in this month, positive for at least one group of tested virus. The correlation between the detection of these different viral agents and environmental factors is discussed. To the knowledge of the authors, this is the first description of viral genomes in water samples taken from the Arroio Dilúvio, Porto Alegre (Brazil).

Adenovírus (AdV), enterovírus (EV), rotavírus (GARV) e Torque teno vírus (TTV) são vírus não envelopados, excretados nas fezes, podendo, assim, contaminar corpos hídricos. No presente estudo, a detecção molecular desses agentes foi realizada em amostras de águas superficiais provenientes do Arroio Dilúvio, o qual cruza a cidade de Porto Alegre-RS, Brasil. As amostras foram coletadas em três meses diferentes (janeiro, abril e setembro) do ano de 2009. A maior taxa de detecção viral foi observada para EV (64,28%), seguida por TTV (28,57%) e AdV (21,43%). Rotavírus não foi detectado. Foi verificada presença simultânea de dois grupos virais em cinco (35,71%) das 14 amostras analisadas. Janeiro foi o mês com a maior taxa de detecção viral, sendo todas as amostras, coletadas nesse mês, positivas para, no mínimo, um grupo viral em estudo. A correlação entre a detecção desses diferentes agentes virais e os fatores ambientais é discutida. Conforme conhecimento dos autores, essa é a primeira descrição de genomas virais em amostras de água provenientes do Arroio Dilúvio, Porto Alegre, Brasil.

Species distribution and antimicrobial susceptibility of enterococci isolated from broilers infected experimentally with Eimeria spp and fed with diets containing different supplements

Resistant bacteria in animal can be spread to environment and to humans. Poultry feed and infections caused by Eimeria spp. are important factors in determining the intestinal microbial communities. The aim of this study was to verify the prevalence of species and antimicrobial susceptibility of Enterococcus isolated from broilers fed with different supplements and infected experimentally with Eimeria spp. Broilers were divided in eight groups, fed with diets supplemented with a combination of antimicrobial, ionophore-coccidiostatics, probiotic, essential oil. At 14 days old all birds, except the control, received a solution containing oocysts of Eimeria spp. Samples of cloacal swabs from broilers were collected. A total of 240 Enterococcus sp. strains were isolated, confirmed genus by PCR, classified as species, tested for antimicrobial susceptibility and screened by PCR for the presence of tet(L), tet(M) and erm(B) genes. The overall distribution of species isolated from fecal samples was E. faecalis (40 percent), followed by E. casseliflavus/E. gallinarum (10.8 percent), E. mundtii (10.8 percent), E. faecium (10.8 percent), E. columbae (5.8 percent) and E. gallinarum (4.2 percent). Changes in the composition or frequency of Enterococcus species were observed in all dietary supplementation. Antimicrobial susceptibility tests showed resistance phenotypes a range of antibiotics, especially used in humans such as, streptomycin, penicillin, rifampicin and vancomycin. There was no correlation between different supplementation for broilers and antimicrobial resistance and the presence of tet(M), tet(L) and erm(B) genes. Dietary supplementation had effect on the Enterococcus sp. colonization, but did not have significant effect on the phenotype and genotype of antimicrobial resistance in enterococci.

The use of RAPD to characterize Bipolaris sorokiniana isolates

Bipolaris sorokiniana is a phytopathogenic fungus causing diseases of cereal crops such as common root rot, the leaf spot disease, seedling blight, and black point of the grain. Random-amplified polymorphic DNA (RAPD) assay was used to investigate the genetic diversity of 20 isolates collected from different cultivars in wheat-producing regions in Brazil. Seventy primers, with random nucleotide sequences, were tested. Reproducibility to amplify the genomic DNA of isolates was found for 30 of the 70 primers tested, generating between 1 and 17 fragments ranging from 0.35 to 2.0 kb (average size). The degree of similarity between samples was calculated through simple association and the dendrogram was assessed using the unweighted pair group method with arithmetical average. After the RAPD analyses 19 isolates were closely grouped, having a similarity coefficient of >or= 78%. Isolate I017 showed very low similarity coefficients, ranging between 38 and 46%. The RAPD analyses provided important information as to the degree of genetic variability and the relationship between the isolates investigated, revealing polymorphism and establishing electrophoretic profiles useful to characterize the phytopathogen.

Intraspecific variability of Bipolaris sorokiniana isolates determined by random-amplified polymorphic DNA (RAPD)

Isolates of Bipolaris sorokiniana were analyzed by random-amplified polymorphic DNA (RAPD) techniques to determine the amount of intraspecific genetic variability and to study host-pathogen interactions. Ten isolates originated from different regions of Brazil were examined. Plants of the wheat cultivars BR8, BH1146 (original host) and IAC-5 Maringá, classified as resistant, moderately resistant or susceptible to B. sorokiniana, respectively, were inoculated with these 10 isolates. Twenty-seven isolates were recovered from these cultivars and were analyzed by RAPD assay and compared to the RAPD of the original 10 isolates. According to the RAPD profiles there was a high level of genetic variability among the isolates. We detected 69 polymorphic fragments, ranging from 1.6 to 0.54 kb, in the original 10 isolates; 57 fragments with sizes between 1.98 and 0.38 kb from the isolates recovered from BH1146; 47 polymorphic bands, ranging from 1.96-0.54 kb, were detected in the isolates from BR8 and 32 fragments between 1.98 and 0.42 kb in isolates were recovered from IAC-5 Maringá. The number of polymorphic fragments varied, even for the same isolate, when the isolates were recovered from different cultivar hosts

Morphological characterization and genetic analysis of Drechslera teres isolates

Net blotch, caused by the phytopathogen Drechslera teres, is a common disease of barley (Hordeum vulgare L) and is responsible for large economic losses in some barley growing areas. In this study the morphology and genetic variability of eight D. teres isolates from different regions of the Brazilian state of Rio Grande do Sul were investigated. Colony morphology was studied on potato-dextrose-agar (PDA) and genetic variability investigated using the random amplified polymorphic-DNA (RAPD) technique. 27 commercially available primers were tested of which 16 were selected for use in polymorphic analysis due to their good resolution and reproducibility. Similarity coefficients were used to construct dendrograms based on colony morphology and RAPD data showing the relationship between the eight isolates studied. Colony morphology showed variability between the isolates while RAPD assays showed high similarity coefficients, but grouping of the isolates according to the geographic origins of the seeds from which they were isolated was not possible