Federal Institute for Risk Assessment

OBJECTIVES: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. METHODS: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. RESULTS: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance. CONCLUSIONS: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.

The polymerase chain reaction (PCR) is increasingly used as the standard method for detection and characterization of microorganisms and genetic markers in a variety of sample types. However, the method is prone to inhibiting substances, which may be present in the analysed sample and which may affect the sensitivity of the assay or even lead to false-negative results. The PCR inhibitors represent a diverse group of substances with different properties and mechanisms of action. Some of them are predominantly found in specific types of samples thus necessitating matrix-specific protocols for preparation of nucleic acids before PCR. A variety of protocols have been developed to remove the PCR inhibitors. This review focuses on the general properties of PCR inhibitors and their occurrence in specific matrices. Strategies for their removal from the sample and for quality control by assessing their influence on the individual PCR test are presented and discussed.

Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 in beef enrichments typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy gene fragments. As these genetic markers can also be found in some non-EHEC strains, a number of "false positive" results are obtained. Here, we explore the suitability of five novel molecular markers, espK, espV, ureD, Z2098, and CRISPRO26:H11 as candidates for a more accurate screening of EHEC strains of greater clinical significance in industrialized countries. Of the 1739 beef enrichments tested, 180 were positive for both stx and eae genes. Ninety (50%) of these tested negative for espK, espV, ureD, and Z2098, but 12 out of these negative samples were positive for the CRISPRO26:H11 gene marker specific for a newly emerging virulent EHEC O26:H11 French clone. We show that screening for stx, eae, espK, and espV, in association with the CRISPRO26:H11 marker is a better approach to narrow down the EHEC screening step in beef enrichments. The number of potentially positive samples was reduced by 48.88% by means of this alternative strategy compared to the European and American reference methods, thus substantially improving the discriminatory power of EHEC screening systems. This approach is in line with the EFSA (European Food Safety Authority) opinion on pathogenic STEC published in 2013.

In this review an overview is given on antibiotic resistance (AR) mechanisms with special attentions to the AR genes described so far preceded by a short introduction on the discovery and mode of action of the different classes of antibiotics. As this review is only dealing with acquired resistance, attention is also paid to mobile genetic elements such as plasmids, transposons, and integrons, which are associated with AR genes, and involved in the dispersal of antimicrobial determinants between different bacteria.

Nanosilver, due to its small particle size and enormous specific surface area, facilitates more rapid dissolution of ions than the equivalent bulk material; potentially leading to increased toxicity of nanosilver. This, coupled with their capacity to adsorb biomolecules and interact with biological receptors can mean that nanoparticles can reach sub-cellular locations leading to potentially higher localized concentrations of ions once those particles start to dissolve or degrade in situ. Further complicating the story is the capacity for nanoparticles to generate reactive oxygen species, and to interact with, and potentially disturb the functioning of biomolecules such as proteins, enzymes and DNA. The fact that the nanoparticle size, shape, surface coating and a host of other factors contribute to these interactions, and that the particles themselves are evolving or ageing leads to further complications in terms of elucidating mechanisms of interaction and modes of action for silver nanoparticles, in contrast to dissolved silver species. This review aims to provide a critical assessment of the current understanding of silver nanoparticle toxicity, as well as to provide a set of pointers and guidelines for experimental design of future studies to assess the environmental and biological impacts of silver nanoparticles. In particular; in future we require a detailed description of the nanoparticles; their synthesis route and stabilisation mechanisms; their coating; and evolution and ageing under the exposure conditions of the assay. This would allow for comparison of data from different particles; different environmental or biological systems; and structure-activity or structure-property relationships to emerge as the basis for predictive toxicology. On the basis of currently available data; such comparisons or predictions are difficult; as the characterisation and time-resolved data is not available; and a full understanding of silver nanoparticle dissolution and ageing under different conditions is observed. Clear concerns are emerging regarding the overuse of nanosilver and the potential for bacterial resistance to develop. A significant conclusion includes the need for a risk—benefit analysis for all applications and eventually restrictions of the uses where a clear benefit cannot be demonstrated.

Bacterial antimicrobial resistance (AMR) is constantly evolving and horizontal gene transfer through plasmids plays a major role. The identification of plasmid characteristics and their association with different bacterial hosts provides crucial knowledge that is essential to understand the contribution of plasmids to the transmission of AMR determinants. Molecular identification of plasmid and strain genotypes elicits a distinction between spread of AMR genes by plasmids and dissemination of these genes by spread of bacterial clones. For this reason several methods are used to type the plasmids, e.g. PCR-based replicon typing (PBRT) or relaxase typing. Currently, there are 28 known plasmid types in Enterobacteriaceae distinguished by PBRT. Frequently reported plasmids [IncF, IncI, IncA/C, IncL (previously designated IncL/M), IncN and IncH] are the ones that bear the greatest variety of resistance genes. The purpose of this review is to provide an overview of all known AMR-related plasmid families in Enterobacteriaceae, the resistance genes they carry and their geographical distribution.

SUMMARY: Throughout much of the world, Trichinella spp. are found to be the causative agents of human trichinellosis, a disease that not only is a public health hazard by affecting human patients but also represents an economic problem in porcine animal production and food safety. Due to the predominantly zoonotic importance of infection, the main efforts in many countries have focused on the control of Trichinella or the elimination of Trichinella from the food chain. The most important source of human infection worldwide is the domestic pig, but, e.g., in Europe, meats of horses and wild boars have played a significant role during outbreaks within the past 3 decades. Infection of humans occurs with the ingestion of Trichinella larvae that are encysted in muscle tissue of domestic or wild animal meat. Early clinical diagnosis of trichinellosis is rather difficult because pathognomonic signs or symptoms are lacking. Subsequent chronic forms of the disease are not easy to diagnose, irrespective of parameters including clinical findings, laboratory findings (nonspecific laboratory parameters such as eosinophilia, muscle enzymes, and serology), and epidemiological investigations. New regulations laying down rules for official controls for Trichinella in meat in order to improve food safety for consumers have recently been released in Europe. The evidence that the disease can be monitored and to some extent controlled with a rigorous reporting and testing system in place should be motivation to expand appropriate programs worldwide.

When Shiga toxin-producing Escherichia coli (STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and stx genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.

Tobacco smoke is a toxic and carcinogenic mixture of more than 5,000 chemicals. The present article provides a list of 98 hazardous smoke components, based on an extensive literature search for known smoke components and their human health inhalation risks. An electronic database of smoke components containing more than 2,200 entries was generated. Emission levels in mainstream smoke have been found for 542 of the components and a human inhalation risk value for 98 components. As components with potential carcinogenic, cardiovascular and respiratory effects have been included, the three major smoke-related causes of death are all covered by the list. Given that the currently used Hoffmann list of hazardous smoke components is based on data from the 1990s and only includes carcinogens, it is recommended that the current list of 98 hazardous components is used for regulatory purposes instead. To enable risk assessment of components not covered by this list, thresholds of toxicological concern (TTC) have been established from the inhalation risk values found: 0.0018 μg day(-1) for all risks, and 1.2 μg day(-1) for all risks excluding carcinogenicity, the latter being similar to previously reported inhalation TTCs.

MicroRNA (miRNA) are a newly recognized class of small, noncoding RNA molecules that participate in the developmental control of gene expression. We have studied the regulation of a set of highly expressed neural miRNA during mouse brain development. Temporal control is a characteristic of miRNA regulation in C. elegans and Drosophila, and is also prominent in the embryonic brain. We observed significant differences in the onset and magnitude of induction for individual miRNAs. Comparing expression in cultures of embryonic neurons and astrocytes we found marked lineage specificity for each of the miRNA in our study. Two of the most highly expressed miRNA in adult brain were preferentially expressed in neurons (mir-124, mir-128). In contrast, mir-23, a miRNA previously implicated in neural specification, was restricted to astrocytes. mir-26 and mir-29 were more strongly expressed in astrocytes than neurons, others were more evenly distributed (mir-9, mir-125). Lineage specificity was further explored using reporter constructs for two miRNA of particular interest (mir-125 and mir-128). miRNA-mediated suppression of both reporters was observed after transfection of the reporters into neurons but not astrocytes. miRNA were strongly induced during neural differentiation of embryonic stem cells, suggesting the validity of the stem cell model for studying miRNA regulation in neural development.

Background and aim Plasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes ( mcr-1 to mcr-5 , and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1 , mcr-2 , mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae . The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.6** , was also identified. Conclusions: This method allows rapid identification of mcr -positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing.

This overview evaluates the capabilities of mass spectrometry (MS) in combination with gas chromatography (GC) and liquid chromatography (LC) for the determination of a multitude of pesticides. The selection of pesticides for this assessment is based on the status of production, the existence of regulations on maximum residue levels in food, and the frequency of residue detection. GC-MS with electron impact (EI) ionization and the combination of LC with tandem mass spectrometers (LC-MS/MS) using electrospray ionization (ESI) are identified as techniques most often applied in multi-residue methods for pesticides at present. Therefore, applicability and sensitivity obtained with GC-EI-MS and LC-ESI-MS/MS is individually compared for each of the selected pesticides. Only for one substance class only, the organochlorine pesticides, GC-MS achieves better performance. For all other classes of pesticides, the assessment shows a wider scope and better sensitivity if detection is based on LC-MS.

The Codex Alimentarius Commission of the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) develops food standards, guidelines and related texts for protecting consumer health and ensuring fair trade practices globally. The major part of the world's population lives in more than 160 countries that are members of the Codex Alimentarius. The Codex Standard on Infant Formula was adopted in 1981 based on scientific knowledge available in the 1970s and is currently being revised. As part of this process, the Codex Committee on Nutrition and Foods for Special Dietary Uses asked the ESPGHAN Committee on Nutrition to initiate a consultation process with the international scientific community to provide a proposal on nutrient levels in infant formulae, based on scientific analysis and taking into account existing scientific reports on the subject. ESPGHAN accepted the request and, in collaboration with its sister societies in the Federation of International Societies on Pediatric Gastroenterology, Hepatology and Nutrition, invited highly qualified experts in the area of infant nutrition to form an International Expert Group (IEG) to review the issues raised. The group arrived at recommendations on the compositional requirements for a global infant formula standard which are reported here.

Lactobacillus acidophilus NCFM is a probiotic bacterium that has been produced commercially since 1972. The complete genome is 1,993,564 nt and devoid of plasmids. The average GC content is 34.71% with 1,864 predicted ORFs, of which 72.5% were functionally classified. Nine phage-related integrases were predicted, but no complete prophages were found. However, three unique regions designated as potential autonomous units (PAUs) were identified. These units resemble a unique structure and bear characteristics of both plasmids and phages. Analysis of the three PAUs revealed the presence of two R/M systems and a prophage maintenance system killer protein. A spacers interspersed direct repeat locus containing 32 nearly perfect 29-bp repeats was discovered and may provide a unique molecular signature for this organism. In silico analyses predicted 17 transposase genes and a chromosomal locus for lactacin B, a class II bacteriocin. Several mucus- and fibronectin-binding proteins, implicated in adhesion to human intestinal cells, were also identified. Gene clusters for transport of a diverse group of carbohydrates, including fructooligosaccharides and raffinose, were present and often accompanied by transcriptional regulators of the lacI family. For protein degradation and peptide utilization, the organism encoded 20 putative peptidases, homologs for PrtP and PrtM, and two complete oligopeptide transport systems. Nine two-component regulatory systems were predicted, some associated with determinants implicated in bacteriocin production and acid tolerance. Collectively, these features within the genome sequence of L. acidophilus are likely to contribute to the organisms' gastric survival and promote interactions with the intestinal mucosa and microbiota.

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 7-9 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 5-7 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

Abstract The flavonoid quercetin is frequently found in low amounts as a secondary plant metabolite in fruits and vegetables. Isolated quercetin is also marketed as a dietary supplement, mostly as the free quercetin aglycone, and frequently in daily doses of up to 1000 mg d –1 exceeding usual dietary intake levels. The present review is dedicated to safety aspects of isolated quercetin used as single compound in dietary supplements. Among the numerous published human intervention studies, adverse effects following supplemental quercetin intake have been rarely reported and any such effects were mild in nature. Published adequate scientific data for safety assessment in regard to the long‐term use (&gt;12 weeks) of high supplemental quercetin doses (≥1000 mg) are currently not available. Based on animal studies involving oral quercetin application some possible critical safety aspects could be identified such as the potential of quercetin to enhance nephrotoxic effects in the predamaged kidney or to promote tumor development especially in estrogen‐dependent cancer. Furthermore, animal and human studies with single time or short‐term supplemental quercetin application revealed interactions between quercetin and certain drugs leading to altered drug bioavailability. Based on these results, some potential risk groups are discussed in the present review.

Structured frameworks are extremely useful in promoting transparent, harmonized approaches to the risk assessment of chemicals. One area where this has been particularly successful is in the analysis of modes of action (MOAs) for chemical carcinogens in experimental animals and their relevance to humans. The International Programme on Chemical Safety (IPCS) recently published an updated version of its MOA framework in animals to address human relevance (cancer human relevance framework, or HRF). This work has now been extended to noncancer effects, with the eventual objective of harmonizing framework approaches to both cancer and noncancer endpoints. As in the cancer HRF, the first step is to determine whether the weight of evidence based on experimental observations is sufficient to establish a hypothesized MOA. This comprises a series of key events causally related to the toxic effect, identified using an approach based on the Bradford Hill criteria. These events are then compared qualitatively and, next, quantitatively between experimental animals and humans. The output of the analysis is a clear statement of conclusions, together with the confidence, analysis, and implications of the findings. This framework provides a means of ensuring a transparent evaluation of the data, identification of key data gaps and of information that would be of value in the further risk assessment of the compound, such as on dose-response relationships, and recognition of potentially susceptible subgroups, for example, based on life-stage considerations.

OBJECTIVES: Plasmid-mediated mobilized colistin resistance is currently known to be caused by phosphoethanolamine transferases termed MCR-1, MCR-2, MCR-3 and MCR-4. However, this study focuses on the dissection of a novel resistance mechanism in mcr-1-, mcr-2- and mcr-3-negative d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B (Salmonella Paratyphi B dTa+) isolates with colistin MIC values >2 mg/L. METHODS: A selected isolate from the strain collection of the German National Reference Laboratory for Salmonella was investigated by WGS and bioinformatical analysis to identify novel phosphoethanolamine transferase genes involved in colistin resistance. Subsequently PCR screening, S1-PFGE and DNA-DNA hybridization were performed to analyse the prevalence and location of the identified mcr-5 gene. Cloning and transformation experiments in Escherichia coli DH5α and Salmonella Paratyphi B dTa+ control strains were carried out and the activity of MCR-5 was determined in vitro by MIC testing. RESULTS: In this study, we identified a novel phosphoethanolamine transferase in 14 mcr-1-, mcr-2- and mcr-3-negative Salmonella Paratyphi B dTa+ isolates with colistin MIC values >2 mg/L that were received during 2011-13. The respective gene, further termed as mcr-5 (1644 bp), is part of a 7337 bp transposon of the Tn3 family and usually located on related multi-copy ColE-type plasmids. Interestingly, in one isolate an additional subclone with a chromosomal location of the mcr-5 transposon was observed. CONCLUSIONS: Our findings suggest that the transfer of colistin-resistance-mediating phosphoethanolamine transferase genes from bacterial chromosomes to mobile genetic elements has occurred in multiple independent events raising concern regarding their variety, prevalence and impact on public health.

A robust 5' nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10(3) CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10(4) CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.

The explosive increase since the beginning of 1990s in the number of publications reporting PCR-based methods for detection or molecular typing of food-borne pathogens has attracted the attention of end-user laboratories. However, the well recognized difficulties in reproducing published tests due