98 Chapter 6 distinguishing of related from non-related plasmids based on molecular characteristics (e.g., number of single nucleotide polymorphisms (SNP) differences) is essential for using sequence data to detect plasmid transmission. We hypothesize that a combination of short- and long-read sequence data of blaCMY-2 containing IncI1–pST12 plasmids reveal highly conserved plasmid sequencing, which complicates distinguishing plasmid transmission between epidemiologically related and unrelated isolates. The objective of the current study is to determine the relatedness between IncI1–pST12 plasmids of epidemiologically related and unrelated Enterobacteriaceae isolates from humans and livestock, and we explore the possibility of accurately distinguishing related from unrelated samples based on plasmid sequencing data alone. Materials and Methods Collection of Isolates AmpC E. coli Isolates from i-4-1-Health Dutch-Belgian Cross-Border Project As part of the i-4-1-Health project, human and broiler samples were collected as described by Kluytmans-van den Bergh et al (Kluytmans-van Den Bergh et al. 2019). After vortexing, the nylon-flocked swabs in 2 mL Cary–Blair medium (FecalSwab®, Copan Italy, Brescia, Italy) were plated on a blood agar plate (growth control, performed since 2011), and the liquid Cary–Blair medium was mixed in tryptic soy broth (TSB) and incubated for 18–24 h (35–37 °C). Broths were subcultured on an AmpC selective MacConkey agar containing cefotaxime and cefoxitin (1 and 8 mg/mL, respectively) on half the plate and ceftazidime and cefoxitin (1 and 8 mg/mL, respectively) on the other half of the plate (Mediaproducts, Groningen, The Netherlands) (Drijver et al. 2019). For all oxidase-negative isolates that grew on either side of the selective agar plates, species identification was performed by automated mass spectrometry systems (VitekMS, bioMérieux, Marcy l’Etoile, France). Susceptibility testing was performed using Vitek 2 (bioMérieux, Marcy l’Etoile, France). The presence of AmpC in all oxidase-negative isolates was phenotypically confirmed using the D68C AmpC & ESBL Detection Set (Mastdiscs, Mastgroup Ltd., Bootle, UK) and interpreted according to the manufacturer’s instructions. All phenotypically confirmed isolates were sequenced using an Illumina MiSeq sequencer (Illumina, San Diego, CA, USA). DNA isolation and sequencing were performed as described by Coolen et al (Coolen et al. 2019). De novo assembly and error correction were performed using SPAdes version 3.9.1 (Bankevich et al. 2012).
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