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
Global dissemination of ciprofloxacin-resistant Salmonella Kentucky has been observed over the past decades. In recent years, there have been reports of extended-spectrum β-lactamase (ESBL) producing S . Kentucky. Routine surveillance at the European Centre for Disease Prevention and Control (ECDC) detected cases with a ciprofloxacin-resistant S . Kentucky with the ESBL-gene bla CTX-M-14b . Ensuing research identified 78 cases in 2013–2018 in eight European countries. Compared to other S . Kentucky and non-typhoidal Salmonella infections, reported to the European Surveillance System, these cases were more likely to be elderly and to present urinary-tract infections. Bayesian time-scaled phylogeny on whole genome sequences of isolates from these cases and supplementary isolates from public sequence databases was used to infer the origin and spread of this clone. We dated the origin of the bla CTX-M-14b clone to approximately 2005 in Northern Africa, most likely in Egypt. The geographic origin predicted by the phylogenetic analysis is consistent with the patients’ travel history. Next to multiple introductions of the clone to Europe from Egypt, our analysis suggests that in some parts of Europe the clone might have formed a stable population, from which further spread has occurred. Comparative genomics indicated that the bla CTX-M-14b gene is present on the bacterial chromosome, within the type VI secretion system region. The bla CTX-M-14b gene is integrated downstream of the hcp1 gene, on a 2854 bp plasmid fragment containing also IS Ecp1 . This is the first report of a chromosomally integrated CTX-M gene in Salmonella spp. in Europe, previous studies having identified similar genes only on plasmids.
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