Rapid and simple universal Escherichia coli genotyping method based on multiple-locus variable-number tandem-repeat analysis using single-tube multiplex PCR and standard gel electrophoresis

Abstract

We developed a multiplex PCR method based on multiple-locus variablenumber tandem-repeat (VNTR) analysis (MLVA) that was designed for the rapid typing of Escherichia coli and Shigella isolates. The method amplifies seven VNTRs and does not require a sequencing capillary or fluorescent dyes. The amplification products are simply loaded on a standard agarose gel for electrophoresis, and the banding patterns are analyzed visually. We evaluated the method on 220 strains belonging to different collections: the E. coli reference (ECOR) collection (n=72), O1:K1 isolates causing neonatal meningitis (n=38), extended-spectrum beta-lactamaseproducing fecal isolates belonging to the worldwide sequence type 131 (ST131) clone (n=38), Shiga toxin-producing E. coli (STEC) isolates of serogroups O157:H7 (n=21) and O26 (n=16, 8 of which belonged to an outbreak), 27 Shigella isolates (22 Shigella sonnei isolates, including 5 epidemic strains), and 8 reference strains. The performances were compared to those of multilocus sequence typing (MLST), the DiversiLab automated repetitive element palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). We found 66 different profiles among the isolates in the ECOR collection. Among the clonal group O1:K1 isolates, 14 different profiles were identified. For the 37 STEC isolates, we found 23 profiles, with 1 corresponding to the 8 epidemic strains. We found 19 profiles among the 27 Shigella isolates, with 1 corresponding to the epidemic strain. The method was able to recognize strains of the ST131 clone and to distinguish the O16 and O25b serogroups and identified 15 different MLVA types among them. This method allows the simple, fast, and inexpensive typing of E. coli/Shigella isolates that can be carried out in any laboratory equipped for molecular biology and has a discriminatory power superior to that of MLST and DiversiLab REP-PCR but slightly lower than that of PFGE.