Browsing Articles and other publications by RIVM employees by Subjects
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The Plasmidome as a Reservoir of Antibiotic Resistance.The emergence of multidrug-resistant bacterial strains worldwide has become a serious problem for public health over recent decades. The increase in antimicrobial resistance has been expanding via plasmids as mobile genetic elements encoding antimicrobial resistance (AMR) genes that are transferred vertically and horizontally. This study focuses on Salmonella enterica, one of the leading foodborne pathogens in industrialized countries. S. enterica is known to carry several plasmids involved not only in virulence but also in AMR. In the current paper, we present an integrated strategy to detect plasmid scaffolds in whole genome sequencing (WGS) assemblies. We developed a two-step procedure to predict plasmids based on i) the presence of essential elements for plasmid replication and mobility, as well as ii) sequence similarity to a reference plasmid. Next, to confirm the accuracy of the prediction in 1750 S. enterica short-read sequencing data, we combined Oxford Nanopore MinION long-read sequencing with Illumina MiSeq short-read sequencing in hybrid assemblies for 84 isolates to evaluate the proportion of plasmid that has been detected. At least one scaffold with an origin of replication (ORI) was predicted in 61.3% of the Salmonella isolates tested. The results indicated that IncFII and IncI1 ORIs were distributed in many S. enterica serotypes and were the most prevalent AMR genes carrier, whereas IncHI2A/IncHI2 and IncA/C2 were more serotype restricted but bore several AMR genes. Comparison between hybrid and short-read assemblies revealed that 81.1% of plasmids were found in the short-read sequencing using our pipeline. Through this process, we established that plasmids are prevalent in S. enterica and we also substantially expand the AMR genes in the resistome of this species.
Selenite enrichment broth to improve the sensitivity in molecular diagnostics of Salmonella.Selenite enrichment broth (SEB) is used to optimize the recovery of Salmonella enterica subspecies enterica from stool samples. Compared to a direct culture approach, it enhances culture yield by reducing growth of faecal coliforms and faecal streptococci. Over the course of seven years from 2000 to 2017, 47,235 faecal samples were tested with a Salmonella PCR. We investigated the added value of using SEB in combination with faeces for DNA extraction, in order to improve the sensitivity of molecular diagnostics for detection of Salmonella. A Salmonella enterica subspecies enterica strain was tested for growth characteristics, with and without incubation in SEB, to determine the impact of Selenite enrichment in the Salmonella PCR. Retrospectively, a total of 102 Salmonella enterica subspecies enterica PCR positive faecal samples were re-analysed. DNA extraction was performed with the EasyMag® and MagNaPure96® system using three different input volumes of faeces and SEB. Prospectively, 114 Salmonella PCR positive faecal samples were retested within 2 days using five different input volumes for DNA extraction. Retrospectively, PCR that used SEB as part of input in the DNA extraction, 7/102 (7%) Salmonella PCR positive samples were additionally detected compared to no use of SEB. Of these, Salmonella enterica subspecies enterica serovariation Thompson, Enteritidis, 9,12:l.v and Senftenberg have been outbreak related in the past. Prospectively results were combined in collaboration with another microbiology laboratory, 15/114 (13.2%) additional specimens were detected with the Salmonella PCR, including processing Selenite enrichment broth. In conclusion, of the total 47,235 feacal samples, with SEB the prevalence of a positive PCR for Salmonella is 2.2%. Of these 2.2% positive Salmonella PCRs, 0.4% was not detected in culture. By using SEB an improved detection of Salmonella diagnostics could be realized and a substantial part of 13,2% additional Salmonella cases could be detected.