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Novel Multiplex PCR Method and Genome Sequence-Based Analog for High-Resolution Subclonal Assignment and Characterization of Escherichia coli Sequence Type 131 Isolates.
Johnston, Brian D; Gordon, David M; Burn, Samantha; Johnson, Timothy J; Weber, Bonnie P; Miller, Elizabeth A; Johnson, James R.
Affiliation
  • Johnston BD; Minneapolis VA Health Care System, Minneapolis, Minnesota, USA.
  • Gordon DM; University of Minnesota, Minneapolis, Minnesota, USA.
  • Burn S; Research School of Biology, The Australian National University Australia, Canberra, Australian Capital Territory, Australia.
  • Johnson TJ; Research School of Biology, The Australian National University Australia, Canberra, Australian Capital Territory, Australia.
  • Weber BP; Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota, USA.
  • Miller EA; Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota, USA.
  • Johnson JR; Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota, USA.
Microbiol Spectr ; 10(3): e0106422, 2022 06 29.
Article in En | MEDLINE | ID: mdl-35604132
Escherichia coli sequence type 131 (ST131) is a pandemic, multidrug-resistant extraintestinal pathogen. The multiple distinctive ST131 subclones differ for rfb and fliC alleles (O and H antigens), fimH allele (type-1 fimbriae adhesin), resistance phenotype and genotype, clinical correlates, and host predilection. Current PCR assays for detecting ST131 and its main subclones offer limited sub-ST characterization. Here we combined 22 novel and 14 published primers for a multiplex PCR assay to detect and extensively characterize ST131 isolates. The primers target mdh36, gyrB47, trpA72, sbmA, plsB, nupC, rmuC, kefC, ybbW, the O16 and O25b rfb variants, five fimH alleles (fimH22, fimH27, fimH30, fimH35, and fimH41), two fliC alleles (H4 and H5), a (subclone-specific) fluoroquinolone resistance-associated parC allele, and a (subclone-specific) prophage marker. The resulting amplicons resolve 15 molecular subsets within ST131, including 3 within clade A (H41 subclone), 5 within clade B (H22 subclone), and 7 within clade C (H30 subclone), which includes subclones C0 (H30S: 2 subsets), C1 and C1-M27 (H30R1: 2 subsets), and C2 (H30Rx: 3 subsets). Validation in three laboratories showed that this assay provides a rapid, accurate, and portable method for rapidly detecting and characterizing E. coli ST131 and its key subsets. Additionally, for users with whole genome sequencing (WGS) capability, we developed a command-line executable called ST131Typer, an in silico version of the extended multiplex PCR assay. Its accuracy was 87.8%, with most issues due to incomplete or fragmented input genome assemblies. These two novel assays should facilitate detailed ST131 subtyping using either endpoint PCR or WGS. IMPORTANCE These novel assays provide greater subclonal resolution and characterization of E. coli ST131 isolates than do the available comparable PCR assays, plus offer a novel sequence-based alternative to PCR. They may prove useful for molecular epidemiological studies, surveillance, and, potentially, clinical management.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Escherichia coli Proteins / Escherichia coli Infections Limits: Humans Language: En Journal: Microbiol Spectr Year: 2022 Document type: Article Affiliation country: United States Country of publication: United States

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Escherichia coli Proteins / Escherichia coli Infections Limits: Humans Language: En Journal: Microbiol Spectr Year: 2022 Document type: Article Affiliation country: United States Country of publication: United States