Antimicrobial resistance (AMR) is a major public health threat. Plasmids are able to transfer AMR genes among bacterial isolates. Whole genome sequencing (WGS) is a powerful tool to monitor AMR determinants. However, plasmids are difficult to reconstruct from WGS data. This study aimed to improve the characterization, including the localization of AMR genes using short and long read WGS strategies. We used a genetically modified (GM) Bacillus subtilis isolated as unexpected contamination in a feed additive, and therefore considered unauthorized (RASFF 2014.1249), as a case study. In GM organisms, AMR genes are used as selection markers. Because of the concern of spread of these AMR genes when present on mobile genetic elements, it is crucial to characterize their location. Our approach resulted in an assembly of one chromosome and one plasmid, each with several AMR determinants of which five are against critically important antibiotics. Interestingly, we found several plasmids, containing AMR genes, integrated in the chromosome in a repetitive region of at least 53 kb. Our findings would have been impossible using short reads only. We illustrated the added value of long read sequencing in addressing the challenges of plasmid reconstruction within the context of evaluating the risk of AMR spread. Antimicrobial resistance (AMR) genes are naturally present in bacteria, where they function as a defense mechanism. However, the overuse of antibiotics in humans and animals over several decades has led to a rapid rise in the prevalence of AMR genes and the emergence of new AMR mechanisms. The monitoring of AMR is of the utmost importance to have an overview of the circulating resistance genes to implement policies to reduce AMR 1,2. The gold standards for AMR detection are phenotypic susceptibility tests and genotypic (q)PCRs. These methods lack the flexibility to continuously search for new mutations/genes and can be very time-consuming. The revolution in DNA-sequencing technologies, i.e. the so-called whole genome sequencing (WGS) technologies combined with specific databases 3 , offers a solution as an efficient, high-throughput analysis method for the characterization of AMR genes. AMR genes can be present on the chromosome or on mobile elements, such as plasmids. Plasmids facilitate the spread of AMR genes, due to their ability to transfer to other bacteria, which is even possible across the species barrier 4,5. Despite the importance of plasmids for the monitoring of AMR genes, it has been shown that they are

No abstract

No abstract

No abstract

To determine the repeatability and reproducibility figures of the AOAC First Action Official MethodSM 2014.02 (Vitamin B12 in Infant Formula and Adult/Pediatric Formula by Liquid Chromatography with UV Detection), a collaborative study was organized. Twenty-one laboratories located in 13 different countries agreed to participate. The study was divided into two parts. During the first part, the laboratories analyzed two samples in duplicate by using the method described in the protocol. The laboratories that provided results within the expected range were qualified for part two, during which they analyzed 10 samples in blind duplicates. Eighteen laboratories managed to provide results on time for reporting. The results were compared with the Standard Method Performance Requirement (SMPR® 2011.005) established for vitamin B12. The precision results met the requirements stated in the SMPR except for one sample. Repeatability and reproducibility relative standard deviation ranged from 1.1 to 6.5% and from 6.0 to 23.8%, respectively, with only one matrix showing reproducibility values higher than the required 11%. Horwitz ratio values were all well below 2 (0.17-0.78). The AOAC Expert Review Panel (Stakeholder Panel for Infant Formula and Adult Nutritional Expert Review Panel) determined that the data presented met the SMPR and, hence, recommended the method to be granted Final Action status in September 2016.