Rapid and Specific Detection of Escherichia coli Sequence Type 131 (ST131) and its Key Subclones Using a Novel Single-tube Multiplex PCR Assay

Abstract

Background E. coli ST131 is a recently emerged, pandemic multidrug-resistant pathogen. ST131 compromises multiple distinctive sub-clones that differ by serotype, fimH (type-1 fimbriae adhesin) allele, resistance phenotype and genotype, and host group predilection. PCR assays have been described for detecting ST131 and several of its subclones, but not all in single reaction.MethodsTo create a single-tube multiplex PCR assay for detecting key ST131 subclones and their associated O types, 2 novel primers were combined with 9 published primers. The primers target allele-specific SNPs in mdh36 and gyrB4 (from multi-locus sequence typing), fimH30, sbmA (transport protein) and rfb O16 and O25b specific nucleotide sequence. The resulting band combinations allow resolution of ST131 per se and of several well established ST131 sub-clones, including ST131 O16 (a.k.a. H41 or clade A) and 4 variants of ST131 O25b, i.e., non-H30 (a.k.a. H22 or clade B), H30 (a.k.a. clade C), H30 non-Rx (a.k.a. H30R1 or clade C2), and H30Rx (a.k.a. clade C2), which can occur with either fimH30 or an alternate fimH allele (typically fimH35). Primers were designed to have a common annealing temperature and were tested on different thermal cyclersResultsThe ST131 multiplex assay identified correctly by subclone (according to the whole genome phylogeny of Price et al.) all 104 ST131 strains (100%) from Price et al. Additionally, in blinded testing of 90 fresh consecutive E. coli clinical isolates, for assigning isolates to ST131 and to its key subclones the new assay yielded 100% concordance with the published single PCR assays that were run in parallel. Assay performance was consistent across thermal cyclers. Figure 1 demonstrates the different unique PCR profiles.Figure 1:PCR product of 5 ST131 sub-clones and 2 non-ST131.ConclusionThis novel ST131 multiplex PCR assay provides a rapid and specific single-step diagnostic tool for efficiently assigning E. coli isolates to ST131 and its key subclones. It should prove useful for epidemiological studies and potentially clinical diagnostics.Disclosures J. R. Johnson, Merck: Grant Investigator, Research grant Grant Investigator, Research grant