CERTIFICATION REPORT The certification of different mass fractions of DP-ØØ4114-3 in maize seed powder Certified Reference Materials ERM®-BF439a, ERM®-BF439b, ERM®-BF439c, ERM®-BF439d and ERM®-BF439e

This report describes the production of a set of Certified Reference Materials (CRMs) ERM BF439a, b, c, d and e, which are certified for their 4114 maize event mass fractions. These materials were produced following ISO Guide 34:2009 and are certified in accordance with ISO Guide 35:2006. The materials are intended for the calibration or quality control of real-time PCR measurements to identify 4114 maize and/or quantify its mass fraction. As with any reference material, they can also be used for establishing control charts or for carrying out validation studies. The CRMs were accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.JRC.D.2-Standards for Innovation and sustainable Developmen

Molecular characterization of Shewanella and Aeromonas isolates associated with spoilage of Common carp (Cyprinus carpio)

Storage in ice is a common way of preserving commercial fish species but some microorganisms can still contaminate and participate in the spoilage of the product; therefore, identification of potential harmful microbes is important. Thirteen colonies were isolated from common carp (Cyprinus carpio) that had been stored in ice, whose phenotypic identification revealed that they belonged to the genera Aeromonas (n = 5) and Shewanella (n = 8). Molecular genotyping with ERIC-PCR showed clonality only among two of the five Aeromonas isolates and for two groups (n = 3; n = 2) of the eight Shewanella isolates. Sequencing the rpoD gene showed that four Aeromonas isolates belonged to the species Aeromonas salmonicida and one to A. sobria. Of the eight Shewanella, seven isolates cluster with Shewanella putrefaciens and one with Shewanella profunda in the 16S rRNA phylogenetic tree. However, analysis of the gyrB gene showed that these eight isolates could constitute a new species closely related to S. baltica. The Shewanella and A. salmonicida isolates produce off-odours and reduce trimethylamine oxide, indicating that they might contribute to the spoilage of the fish.Fil: Beaz Hidalgo, Roxana. Universitat Rovira I Virgili; EspañaFil: Agüeria, Daniela Alejandra. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Exactas. Instituto Multidisciplinario de Ecosistemas y Desarrollo Sustentable; ArgentinaFil: Latif Eugenín, Fadua. Universitat Rovira I Virgili; EspañaFil: Yeannes, Maria Isabel. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Figueras, Maria J.. Universitat Rovira I Virgili; Españ

Strategies to avoid wrongly labelled genomes using as example the detected wrong taxonomic affiliation for aeromonas genomes in the GenBank database.

Around 27,000 prokaryote genomes are presently deposited in the Genome database of GenBank at the National Center for Biotechnology Information (NCBI) and this number is exponentially growing. However, it is not known how many of these genomes correspond correctly to their designated taxon. The taxonomic affiliation of 44 Aeromonas genomes (only five of these are type strains) deposited at the NCBI was determined by a multilocus phylogenetic analysis (MLPA) and by pairwise average nucleotide identity (ANI). Discordant results in relation to taxa assignation were found for 14 (35.9%) of the 39 non-type strain genomes on the basis of both the MLPA and ANI results. Data presented in this study also demonstrated that if the genome of the type strain is not available, a genome of the same species correctly identified can be used as a reference for ANI calculations. Of the three ANI calculating tools compared (ANI calculator, EzGenome and JSpecies), EzGenome and JSpecies provided very similar results. However, the ANI calculator provided higher intra- and inter-species values than the other two tools (differences within the ranges 0.06-0.82% and 0.92-3.38%, respectively). Nevertheless each of these tools produced the same species classification for the studied Aeromonas genomes. To avoid possible misinterpretations with the ANI calculator, particularly when values are at the borderline of the 95% cutoff, one of the other calculation tools (EzGenome or JSpecies) should be used in combination. It is recommended that once a genome sequence is obtained the correct taxonomic affiliation is verified using ANI or a MLPA before it is submitted to the NCBI and that researchers should amend the existing taxonomic errors present in databases

Intra and inter-species ANI values for the 44 <i>Aeromonas</i> genomes and identification on the basis of the MLPA and ANI (mislabeled genomes in bold).

<p>Intra and inter-species ANI values for the 44 <i>Aeromonas</i> genomes and identification on the basis of the MLPA and ANI (mislabeled genomes in bold).</p

Genome-driven evaluation and redesign of PCR tools for improving the detection of virulence-associated genes in aeromonads.

Many virulence factors have been described for opportunistic pathogens within the genus Aeromonas. Polymerase Chain Reactions (PCRs) are commonly used in population studies of aeromonads to detect virulence-associated genes in order to better understand the epidemiology and emergence of Aeromonas from the environment to host, but their performances have never been thoroughly evaluated. We aimed to determine diagnostic sensitivity and specificity of PCR assays for the detection of virulence-associated genes in a collection of Aeromonas isolates representative for the genetic diversity in the genus. Thirty-nine Aeromonas strains belonging to 27 recognized species were screened by published PCR assays for virulence-associated genes (act, aerA, aexT, alt, ascFG, ascV, ast, lafA, lip, ser, stx1, stx2A). In parallel, homologues of the 12 putative virulence genes were searched from the genomes of the 39 strains. Of the 12 published PCR assays for virulence factors, the comparison of PCR results and genome analysis estimated diagnostic sensitivities ranging from 34% to 100% and diagnostic specificities ranged from 71% to 100% depending upon the gene. To improve the detection of virulence-associated genes in aeromonads, we have designed new primer pairs for aerA/act, ser, lafA, ascFG and ascV, which showed excellent diagnostic sensitivity and specificity. Altogether, the analysis of high quality genomic data, which are more and more easy to obtain, provides significant improvements in the genetic detection of virulence factors in bacterial strains

Determination of GM soybean 44406 in soya milk and GM maize VCO-1981 in maize flour

The European Union Reference Laboratory for Genetically Modified Food and Feed (EURL GMFF) organised a comparative test (CT) for National Reference Laboratories (NRLs) to support the official controls on food and feed in line with Regulation (EC) No 882/2004. Other official control laboratories were allowed to participate on a voluntary basis. Two test items were distributed: a soya milk powder spiked with soybean GM event DAS-444Ø6-6 (Test Item 1, T1) and a maize flour containing maize event VCO-Ø1981-5 (Test Item 2, T2). Participants were required to screen T1 and T2 for the presence of three GM soybean events and three GM maize events, respectively, and to quantify those events identified. The results had to be reported in GM mass/mass %. Eighty-three participants from 38 countries participated to this CT round, including 55 NRLs, of which 33 are designated in line with Regulation (EC) No 882/2004 (NRL/882) and 22 are nominated in Regulation (EU) No 120/2014 to support the EURL GMFF on method validation (NRL/120). The qualitative results, i.e. the correct identification of the GM events, were evaluated and scored as correct or incorrect. The assigned value for the 44406 soybean mass fraction in the soy milk material was derived as the robust mean of the data provided by NRLs, while for VCO-1981, the certified value was set as the assigned value. z and ζ scores were calculated to assess laboratory performance. The results reported indicate that all NRLs identified the correct GM events in both test items and most of the quantitative results were satisfactorily. Five laboratories, including one NRL/882, obtained an unsatisfactory z score for the quantification of 44406 soybean in soya milk powder. All z scores for VCO-1981 maize content in maize flour were satisfactory. A total of 19 and 7 unsatisfactory ζ scores were obtained for events 44406 and VCO-1981, respectively. At least for a number of laboratories this was the due to an underestimation or overestimation of the measurement uncertainty or a failure to report it. A root-cause analysis will be requested from NRLs having reported unsatisfactory results in this CT round and will be followed-up.JRC.F.5-Food and Feed Complianc

Aeromonas aquatica sp. nov., Aeromonas finlandiensis sp. nov. and Aeromonas lacus sp. nov. isolated from Finnish waters associated with cyanobacterial blooms

Three groups of Aeromonas strains isolated from Finland lakes experiencing cyanobacterial blooms could not be assigned to any known species of this genus on the basis of 16S rRNA and rpoD gene sequences. The Multilocus Phylogenetic Analysis (MLPA) of the concatenated sequence of seven genes (gyrB, rpoD, recA, dnaJ, gyrA, dnaX and atpD; 4093bp) showed that the three groups of strains did not cluster with any known Aeromonas spp. and formed three independent lineages. This was confirmed by performing the analysis with their closest relatives using 15 genes (the latter 7 and cpn60, dnaK, gltA, mdh, radA, rpoB, tsf, zipA; 8751bp). Furthermore, ANI results between the genomes of the type strains of the three potential new species and those of their close relatives were all &lt;96% which is the previously proposed cutoff value for differentiating species within this genus. The in silico DDH values of the three type strains of the new species also showed a similarity &lt;70% with the most closely related species indicating they belong to different taxa. The three groups of strains could be differentiated from each other and from other known Aeromonas species on the basis of several phenotypic characters. This polyphasic study revealed that the 3 groups of strains represent 3 novel Aeromonas species for which the names Aeromonas aquatica sp. nov. (type strain AE235T=CECT 8025T=LMG 26712T), Aeromonas finlandiensis sp. nov. (type strain 4287DT=CECT 8028T=LMG 26709T) and Aeromonas lacus sp. nov. (type strain AE122T=CECT 8024T=LMG 26710T) are proposed. © 2015 Elsevier GmbH

First Record of the Rare Species <i>Aeromonas lusitana</i> from Rainbow Trout (<i>Oncorhynchus mykiss</i>, Walbaum): Comparative Analysis with the Existing Strains

The species Aeromonas lusitana was first described in 2016 with five strains recovered from untreated water and vegetables from Portugal. Since then, no further records exist of this species. During a surveillance study on the presence of Aeromonas in fish farms in Mexico, a new strain (ESV-351) of the mentioned species isolated from a rainbow trout was recovered. It was identified because it clustered phylogenetically with the type strain of A. lusitana based on the analysis of the rpoD gene sequences. In the present study, phenotypic characteristics, antimicrobial resistance profiles, and the presence of putative virulence genes of this novel strain (ESV-351) were determined in parallel to the five isolates from the original species description. Phenotypic differential characteristics exhibited by A. lusitana ESV-351 depicted an evident similarity to the characteristics exhibited by the other evaluated strains. However, the novel strain was positive for the production of indole using conventional methods, while the rest of the strains, including the type strain, were negative for its production. Furthermore, intermediate resistance to ampicillin, amoxicillin-clavulanic acid and cephalothin was detected in both the novel and the type strain. Five different virulence-related genes were detected in the novel strain and in the previously described strains, with the type strain exhibiting the highest number of virulence-related genes. In addition to this, the genome of the novel strain (ESV-351) was sequenced and compared with the genomes from the type strain (A. lusitana CECT 7828T) and other Aeromonas spp. The genomic analysis defined Aeromonas tecta as the closest species to A. lusitana with a highly similar number of predicted proteins. The genomic size, the number of protein-encoding genes and the number of different tRNAs, among other characteristics, make it possible to propose that the ESV-351 strain could potentially have the capacity to adapt to different environments. Genome comparison of the ESV-351 strain with the type strain revealed that both possess a similar sequence of the citrate synthase gene. In addition to this finding, the chromosomal region containing the citrate synthase locus of the novel strain exhibits some similarity to the chromosomal region in the genome of the A. hydrophila type strain and other known human pathogens, such as Vibrio cholerae. This could suggest a possible virulence role for the citrate synthase gene in A. lusitana (ESV-351)