Genomic Diversity of Campylobacter lari Group Isolates from Europe and Australia in a One Health Context.

Members of the Campylobacter lari group are causative agents of human gastroenteritis and are frequently found in shellfish, marine waters, shorebirds, and marine mammals. Within a One Health context, we used comparative genomics to characterize isolates from a diverse range of sources and geographical locations within Europe and Australia and assess possible transmission of food, animal, and environmental isolates to the human host. A total of 158 C. lari isolates from Australia, Denmark, France, and Germany, which included 82 isolates from human stool and blood, 12 from food, 14 from domestic animal, 19 from waterbirds, and 31 from the environment were analyzed. Genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance (AMR) traits was carried-out. Most of the isolates belonged to C. lari subsp. lari (Cll; 98, 62.0%), while C. lari subsp. concheus and C. lari urease-positive thermotolerant Campylobacter (UPTC) were represented by 12 (7.6%) and 15 (9.5%) isolates, respectively. Furthermore, 33 (20.9%) isolates were not assigned a subspecies and were thus attributed to distant Campylobacter spp. clades. Whole-genome sequence-derived multilocus sequence typing (MLST) and core-genome MLST (cgMLST) analyses revealed a high genetic diversity with 97 sequence types (STs), including 60 novel STs and 14 cgMLST clusters (≤10 allele differences), respectively. The most prevalent STs were ST-21, ST-70, ST-24, and ST-58 (accounting for 13.3%, 4.4%, 3.8%, and 3.2% of isolates, respectively). A high prevalence of the 125 examined virulence-related loci (from 76.8 to 98.4% per isolate) was observed, especially in Cll isolates, suggesting a probable human pathogenicity of these strains. IMPORTANCE Currently, relatedness between bacterial isolates impacting human health is easily monitored by molecular typing methods. These approaches rely on discrete loci or whole-genome sequence (WGS) analyses. Campylobacter lari is an emergent human pathogen isolated from diverse ecological niches, including fecal material from humans and animals, aquatic environments, and seafood. The presence of C. lari in such diverse sources underlines the importance of adopting an integrated One Health approach in studying C. lari population structure for conducting epidemiological risk assessment. This retrospective study presents a comparative genomics analysis of C. lari isolates retrieved from two different continents (Europe and Australia) and from different sources (human, domestic animals, waterbirds, food, and environment). It was designed to improve knowledge regarding C. lari ecology and pathogenicity, important for developing effective surveillance and disease prevention strategies.

Evolutionary classification of tumor- and root-inducing plasmids based on T-DNAs and virulence regions.

Tumor-inducing (Ti) and root-inducing (Ri) plasmids of Agrobacterium that display a large diversity are involved in crown gall and hairy root plant diseases. Their phylogenetic relationships were inferred from an exhaustive set of Ti and Ri plasmids (including 36 new complete Ti plasmids) by focusing on T-DNA and virulence regions. The opine synthase gene content of T-DNAs revealed 13 opine types corresponding to former classifications based on opines detected in diseased plants, while the T-DNA gene content more finely separate opine types in 18 T-DNA organizations. This classification was supported by the phylogeny of T-DNA oncogenes of Ti plasmids. The five gene organizations found in Ti/Ri vir regions was supported by the phylogeny of common vir genes. The vir organization was found to be likely an ancestral plasmid trait separating "classic" Ti plasmids (with one or two T-DNAs) and "Ri and vine-Ti" plasmids. A scenario generally supported by the repABC phylogeny. T-DNAs likely evolved later with the acquisition of opine characteristics as last steps in the Ti/Ri plasmid evolution. This novel evolutionary classification of Ti/Ri plasmids was found to be relevant for accurate crown gall and hairy root epidemiology.

Pathogenicity, Phylogenetic relationship and NGS based identification and assembly of tumorigenic Agrobacterium radiabacter plasmidic and chromosomic reads isolated from Prunus duclcis.

Although many Agrobacterium radiobacter strains have already been identified, only a few genomes of strains belonging to genomovar G4 have been sequenced so far. In this study, we report the first virulent genome sequence of Agrobacterium radiobacter strain tun 183, which is highly virulent to almond specie. The genome size was estimated to be 5.53 Mb, with 57.9%GC content. In total, 6486 genes encoding proteins and 61 genes encoding RNAs were identified in this genome. Comparisons with the available sequenced genomes of genomovar G4 as well as with other A. sp. were conducted, revealing a hexapartite genome containing circular and linear chromosomes in addition to two accessory plasmids and a tumor inducing plasmid (pTi) in strain tun 183. The phylogenetic analysis of recA gene clearly showed the clustering of tun 183 strain within genomovar G4, supporting the monophyly within this genomovar.

A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria.

The question asked in the present work was how to differentiate between contamination of field samples with and GM plants contained sequences provided from this bacterium in order to avoid false positives in the frame of the detection and the quantification of GMO. For this, new set of primers and corresponding TaqMan Minor Groove Binder (MGB) probes were designed to target Agrobacterium sp. using the tumor-morphology-shooty gene (TMS1). Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log (colony forming units) per milliliter) via linear regression. The method designed was highly specific and sensitive, with a detection limit of 10CFU/ml. No significant cross-reaction was observed. Results from this study showed that TaqMan real-time PCR, is potentially an effective method for the rapid and reliable quantification of Agrobacterium sp. in samples containing GMO or non GMO samples.

A new specific reference gene based on growth hormone gene (GH1) used for detection and relative quantification of Aquadvantage® GM salmon (Salmo salar L.) in food products.

Genetic transformation of fish is mainly oriented towards the improvement of growth for the benefit of the aquaculture. Actually, Atlantic salmon (Salmo salar) is the species most transformed to achieve growth rates quite large compared to the wild. To anticipate the presence of contaminations with GM salmon in fish markets and the lack of labeling regulations with a mandatory threshold, the proper methods are needed to test the authenticity of the ingredients. A quantitative real-time polymerase chain reaction (QRT-PCR) method was used in this study. Ct values were obtained and validated using 15 processed food containing salmon. The relative and absolute limits of detection were 0.01% and 0.01 ng/µl of genomic DNA, respectively. Results demonstrate that the developed QRT-PCR method is suitable specifically for identification of S. salar in food ingredients based on the salmon growth hormone gene 1 (GH1). The processes used to develop the specific salmon reference gene case study are intended to serve as a model for performing quantification of Aquadvantage® GM salmon on future genetically modified (GM) fish to be commercialized.

A new dual plasmid calibrator for the quantification of the construct specific GM canola Oxy-235 with duplex real-time PCR.

To overcome the difficulties of obtaining the Certified Reference Material (CRM) and according to the key documents of the European Union Reference Laboratory (EU-RL), a new standard reference molecule containing the construct specific of the canola event Oxy-235 (3'-junction Nitrilase/Tnos) and the canola endogenous reference gene (acety-CoA-carboxylase) was constructed and used for duplex real-time quantitative analysis. The limits of detection (LOD) were less than 5 Haploid Genome Copy (HGC) and the limits of quantification (LOQ) were about 10 HGC. Furthermore, mixed GM and non-GM canola samples were analysed with duplex QRT-PCR to evaluate the performance criteria as required for validation procedures in the EU-RL, namely, the precision and the accuracy. The accuracy expressed as bias ranged from 2% to 10% and the precision (repeatability and reproducibility) expressed as the RSDr and RSDR was from 2.2 to 5.12 and 2.15 to 5.46 respectively. All these indicated that the developed construct specific method and the reference molecule are suitable for the identification and the quantification of the canola event Oxy-235.

Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet "Beta vulgaris L.": GMO application.

KEY MESSAGE : Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R (2) > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.