Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

<p>Abstract</p> <p>Background</p> <p>Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium <it>Dichelobacter nodosus</it>. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but <it>D. nodosu</it>s should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of <it>D. nodosus </it>and to compare its performance with culturing and conventional PCR.</p> <p>Methods</p> <p>A <it>D. nodosus-</it>specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly.</p> <p>Results</p> <p>The developed assay had a detection limit of 3.9 fg of <it>D. nodosus </it>genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the <it>D. nodosus </it>genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR.</p> <p>Conclusions</p> <p>The developed real-time PCR assay has good specificity and sensitivity for detection of <it>D. nodosus</it>, and the results are easy to interpret. The method is less time-consuming than either culturing or conventional PCR.</p

Plasmid-associated antimicrobial resistance and virulence genes in Escherichia coli in a high arctic reindeer subspecies

publishedVersio

Successful host adaptation of IncK2 plasmids

book of abstracts, pas d'ISBNInternational audienceAntimicrobial resistance is a global health threat and because it is often encoded on plasmids, it is crucial to understand the dynamics of plasmid spread and host adaptation. It was shown that IncK plasmids can be divided into two separate lineages named IncK1 and IncK2. IncK2 plasmids are found predominantly in poultry. The relatively high body temperature of chicken influences IncK2 plasmids fitness cost, copy number and stress response in the Escherichia coli host. These data shed a light on IncK2 plasmid’s success and persistence in E. coli of chicken origin.This study analyzed 50 IncK2 carrying isolates of human, poultry, cattle, pig and environmental origin from 10 European countries and Lebanon, as well as 14 publicly available IncK2 plasmid sequences. IncK2 carrying isolates analyzed in this study were sequenced using both Illumina and Nanopore technology. A phylogenetic analysis of all plasmids was performed in order to determine the genetic relatedness of IncK2 plasmids isolated in different countries and from different sources. Additionally, a genome wide association study (GWAS) was performed on annotated sequences to assess if an association exists between specific genetic features and various sources, that could explain the suspected IncK2 plasmid adaptation to the chicken host. The obtained results show that an antitoxin for the Hok/Gef family protein and YdeA protein were predominantly found on plasmids isolated from chicken and therefore are significantly associated with IncK2 from the chicken host. Moreover, protein YffA is significantly associated with IncK2 from human.In combination with prior findings, this study shows that adaptation of plasmids to a chicken host is a complex process that involves both physiological and genetic determinants. Understanding the basis of plasmid adaptation may lead to the development of intervention strategies that reduce the spread of AMR plasmids between different sources